,, 倪征, 余斌, 华炯钢, 叶伟成, 云涛, 刘可姝, 朱寅初, 张存,浙江省农业科学院畜牧兽医研究所,杭州 310021Optimized Promoter Regulating of Duck Tembusu Virus E Protein Expression Delivered by a Vectored Duck Enteritis Virus in vitro
CHEN Liu,, NI Zheng, YU Bin, HUA JiongGang, YE WeiCheng, YUN Tao, LIU KeShu, ZHU YinChu, ZHANG Cun,Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021通讯作者:
责任编辑: 林鉴非
收稿日期:2020-01-17接受日期:2020-07-29网络出版日期:2020-12-16
| 基金资助: |
Received:2020-01-17Accepted:2020-07-29Online:2020-12-16
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陈柳,Tel：0571-86404257;E-mail：
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陈柳, 倪征, 余斌, 华炯钢, 叶伟成, 云涛, 刘可姝, 朱寅初, 张存. 重组鸭瘟病毒载体中筛选高效表达鸭坦布苏病毒E蛋白启动子[J]. 中国农业科学, 2020, 53(24): 5125-5134 doi:10.3864/j.issn.0578-1752.2020.24.015
CHEN Liu, NI Zheng, YU Bin, HUA JiongGang, YE WeiCheng, YUN Tao, LIU KeShu, ZHU YinChu, ZHANG Cun.
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0 引言
【研究意义】自从2010年发生以来,鸭坦布苏病毒（DTMUV）给我国养鸭业带来了巨大的经济损失。因为其黄病毒属性,鸭坦布苏病毒及其他的水禽TMUV可能纳入公共健康关注。因此,关于DTMUV疫苗的研制是非常迫切的。目前,已有商品化的DTMUV灭活疫苗（HB株）和减毒疫苗（WF100株）。这些疫苗各有优缺点,灭活苗安全性高、但价格高,而减毒疫苗存在一定安全隐患。鸭瘟是鸭、鹅和其他雁形目禽类的一种急性、热性、败血性传染病。该病流行广泛,传播迅速,发病率和死亡率都高,曾给禽类养殖业带来了巨大的经济损失。鸭瘟的防控目前主要靠传统鸭瘟鸡胚化弱毒活疫苗,在水禽生产上广泛应用,具有免疫效果好、安全性高的特点,还能突破母源抗体的干扰。但不能区分免疫鸭和自然感染鸭,影响该病的免疫检测及监控。这也是疫苗研究工作者急需解决的问题。分子生物学技术的发展,疫苗的研究出现了根本性的变化,目前研发的新型的疫苗主要有基因疫苗、亚单位疫苗、合成肽疫苗和载体疫苗等。重组载体疫苗除具有弱毒疫苗的优点外,还具有遗传标记易区别于野毒、容易研发新型多联多价疫苗的特点,深受研究者的青睐,成为新型兽用疫苗研究的热点。【前人研究进展】目前,痘病毒、腺病毒、疱疹病毒、慢病毒、腺联相关病毒已被广泛用于疫苗载体的研发[1,2,3,4,5,6,7,8,9,10,11]。鸭瘟病毒（DEV）除了具有疱疹病毒特性之外,商品化的DEV弱化疫苗株已被广泛用于DEV的防控。因此,DEV是极佳的用于研发水禽疫苗的病毒载体候选者。迄今为止,研究者已尝试以DEV为载体,分别将传染性法氏囊病病毒、鸭病毒性肝炎病毒I型和III型、新城疫病毒、H5N1亚型禽流感病毒（AIV）、H5N6亚型AIV、H5N8亚型AIV、H9N2亚型AIV、DTMUV、鹅细小病毒、鹅H5亚型禽流感病毒的抗原基因插入至DEV基因组中,用于研发相关的二联疫苗[12,13,14,15,16,17,18,19,20,21,22,23,24,25,26]。CHEN等分别将前缀信号肽tPAS的DTMUV截短形式的E蛋白（TE）、和与prM串联表达的E截短体（prM/TE）插入到DEV疫苗株基因组的US7/US8间,获得了两株重组鸭瘟病毒,该病毒感染鸡胚成纤维细胞能表达外源蛋白,并能诱导鸭体产生E蛋白抗体,经过两次免疫,rDEV-PrM/TE能完全保护鸭免受DTMUV的攻击,而rDEV-TE只能保护部分鸭子遭受DTMUV攻毒。该研究显示,tTPA信号肽的加入增加了E蛋白的表达量及分泌型蛋白的产生[24]。ZOU等以DEV作为载体表达DTMUV全长E基因也取得了成功。重组病毒C-KCE-E能保护鸭免于DEV强毒攻击,且能产生DTMUV E蛋白中和性抗体[25]。之后,ZOU等又同时将H5N1亚型禽流感病毒 HA和DTMUV prM-E克隆至DEV疫苗株基因组,获得了重组病毒C-KCE-HA/ PrM-E,该病毒能诱导部分鸭产生针对H5N1亚型AIV和DTMUV的中和性抗体,但同时也能诱导机体产生体液免疫,接种重组病毒能保护鸭免于DEV、H5N1亚型AIV和DTMUV强毒的攻击[15]。虽然这些研究取得了不错的成绩,但没有系统性地研究影响外源E基因在重组DEV载体中的因素,前期我们摸索了DTMUV的抗原形式（包括前缀信号肽的E基因、去除跨膜区的截短的E基因）,并对外源抗原密码子进行优化,选中了一种较为优化的抗原形式E451-dk (简称为Es)。【本研究切入点】虽然课题组已针对DTMUV E蛋白进行了多方面的优化研究,但对于调控蛋白表达的关键因素——启动子这部分工作还没有开展。外源基因序列及调控表达的启动子等对于调控外源基因的表达至关重要。LI等以马立克氏病毒MDV作为活病毒载体表达了传染性法氏囊病病毒IBDV的VP2蛋白,发现在Pec启动子调控下的VP2表达量较pCMV调控下的高,且rDEV-Pec-VP2产生的免疫保护能力较rDEV-CMV-VP2更好,说明重组MDV疫苗对IBDV的保护效率与该病毒外源蛋白VP2的表达水平密切相关[27]。【拟解决的关键问题】为了控制和预防鸭坦布苏病毒感染,本研究拟在前期工作的基础上,通过更换调控外源E451-dk（简称为Es）蛋白表达的启动子,从而筛选出一株以鸭瘟病毒疫苗株作为载体Es基因更高效表达的重组鸭瘟病毒,期望获得一株可以诱导鸭产生DEV和DTMUV两种病毒中和抗体和对病毒产生免疫保护作用的重组鸭瘟病毒,为研究鸭瘟病毒和鸭坦布苏病毒二联活疫苗奠定基础。1 材料与方法
试验于2016年6月至2017年10月在浙江省农业科学院畜牧兽医研究所完成。1.1 菌株、质粒和病毒
pCDNA3.1+、pCAGGS-NLS-Cre质粒由浙江省农业科学院畜牧兽医研究所保存;pBS448 RSV-gfp-Cre质粒购自Addgene公司;pDEV-EF1/GS1783菌株（为携带有鸭瘟病毒疫苗株感染性BAC克隆的菌株）和对应的rDEV-EF1病毒株由浙江省农业科学院畜牧兽医研究所禽病组研究室构建并保存[28,29];携带以鸭为宿主进行密码子优化的DTMUV E451基因（简称为Es）的重组质粒pEP-BGH-E451-dk（即pEP-BGH-Es）和重组鸭瘟病毒rDEV-Es（即rDEV-pCMV-Es）由浙江省农业科学院畜牧兽医研究所禽病组研究室构建并保存[30]。MDV RISPEN疫苗株购自乾元浩生物股份有限公司。1.2 主要试剂
KOD酶购自东洋纺（上海）生物科技有限公司;限制性内切酶、凝胶纯化试剂盒、快速连接试剂盒、Western BLoT Chemiluminescence HRP Substrate化学发光底物购自于宝生物工程（大连）有限公司;质粒提取试剂盒为Omega公司产品;Trans-T1化学感受态细胞、蛋白分子量Marker（EasySee Western Marker）购自于北京全式金生物技术有限公司;胎牛血清为Gibco BRL公司产品;磷酸钙转染试剂盒为Promega公司产品;DMEM高糖培养基购自于上海吉诺生物医药有限公司;酶标二抗（HRP标记的山羊抗小鼠IgG、HRP标记的山羊抗兔IgG）购于Santa Cruz公司;GFP单抗购于碧云天生物技术公司。蛋白浓缩超滤管（30k）购于millipore公司;小鼠抗鸡beta-actin单克隆抗体购自Sigma。1.3 细胞
取9—11日龄SPF鸡胚采用胰蛋白酶消化法[31]制备鸡胚成纤维细胞（CEFs）。SPF鸡胚由浙江省农业科学院良种家禽孵化基地提供。1.4 TUMV E蛋白DIII结构域多克隆抗体的制备
TUMV E蛋白DⅢ结构域抗原由本课题组制备[32],按常规方法免疫兔制备兔抗DIII多克隆抗体,以纯化的DIII蛋白作为抗原包被酶标板,采用间接ELISA方法测定抗体效价。DEV UL44多克隆抗体制备方法同上,采用原核表达法制备抗原,所用引物为Dev UL44（BamHI+）和Dev UL44 （XhoI-）引物对（表1）。纯化抗原免疫小鼠制备鼠抗UL44多克隆抗体,采用间接ELISA方法测定抗体效价。Table 1
表1
表1本文所用引物
Table 1
| 引物名称 Primer | 序列 Sequence | 引入位点 Sequence introduced |
|---|---|---|
| pCAG(MluI+) | 5′-cgACGCGTTAGTTATTAATAGTAATCAATTACG-3′ | Mlu I |
| PCAG(NheI-) | 5′-ctaGCTAGCGCCGCCGGTCACACGCCAGAAGCC-3′ | Nhe I |
| pRSV(BglII+) | 5′-gaAGATCTCTGCTCCCTGCTTGTGTGTTG-3′ | Bgl II |
| pRSV(NheI-) | 5′-ctaGCTAGCGTGCACACCAATGTGGTGAATG-3′ | Nhe I |
| pSV40(MluI+) | 5′-cgACGCGTCTGTGGAATGTGTGTCAGTTAGG-3′ | Mlu I |
| pSV40(NheI-) | 5′-ctaGCTAGCCGAAAATGGATATACAAGCTCCCGG-3′ | Nhe I |
| F(MDV p1.8k BglII+) | 5′-gaAGATCTTCGAGGCCACAAGAAATTAC-3′ | Bgl II |
| R(MDV p1.8k NheI-) | 5′-ctaGCTAGCGAGCATCGCGAAGAGAGAAG-3′ | Nhe I |
| F(MDV gB BglII+) | 5'-cgAGATCTCAAGTCTCACTCACAAATTTTTTC-3′ | Bgl II |
| R(MDV gB NheI-) | 5′-ctaGCTAGCAGTGAGATGATCTTAATGATGC-3′ | Nhe I |
| pDEV vac-in-s(p1.8k,pgB) | 5′-TACTAATTTAAGTGTGCAGCCTGGTTAACTGTATTATGCGCGGAGTGACGTCGACGGATCGGG-3′ | |
| pDEV vac-in-s | 5′-TACTAATTTAAGTGTGCAGCCTGGTTAACTGTATTATGCGCGGAGCGATGTACGGGCCAGATA-3′ | |
| pDEV vac-in-as | 5′- TCCGTAGTCTGGCCGGCAGTATGTTGGTGTTTAGTACTCCAAACCCA TAGAGCCCACCGCATCCCC-3′ | |
| JD-F | 5′-CTACCACAAGCGTCATCAACCA-3′ | |
| JD-R | 5′-TGTCCATTACCAAATCCGAAAA-3′ | |
| DEV-tk-F | 5′- GCTTCCCAGCAGCTCGTT-3′ | |
| DEV-tk-R | 5′- TCTCGTACTTCAGCGGCACA-3′ | |
| Dev UL44(BamHI+) | 5′-cgGGATCCATGGGGCCATTAGTGATGGTTG-3′ | BamH I |
| Dev UL44 (XhoI-) | 5′-ccgCTCGAGTCAAATAATATTGTCTGCTTTATC-3′ | XhoI |
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1.5 引物设计及合成
本文所用引物见表1：pCAG（MluI+）/ PCAG （NheI-）引物对用于以pCAGGS-NLS-Cre为模板扩增pCAG基因;pRSV（BglII+）/pRSV（NheI-）用于以pBS448 RSV-gfp-Cre质粒为模板扩增pRSV基因;pSV40（MluI+）/pSV40（（NheI-）用于以pCDNA3.1+为模板扩增pSV40基因;F（MDV p1.8k BglII+）/R（MDV p1.8k NheI-）用于以MDV RISPEN病毒DNA为模板扩增MDV 1.8k基因启动子（p1.8k）;F（MDV gB BglII+）/ R（MDV gB NheI-）用于以MDV RISPEN病毒DNA为模板扩增MDV gB启动子（pgB）。pDEV vac-in-s和pDEV vac-in-as用于分别将表达框pCAG- Es-BGH-pA、pRSV-Es-BGH-pA和pSV40-Es-BGH-pA插入到DEV基因组的US7和US8基因之间;pDEV vac-in-s（p1.8k,pgB）和pDEV vac-in-as用于分别将表达框p1.8k（MDV）-Es-BGH-pA、pgB（MDV）-Es-BGH- pA插入到DEV基因组的US7和US8基因之间。下划线部分与pEP-pro-Es上序列同源,可以从pEP-pro-Es扩增出含有外源基因表达框及Kan筛选基因的片段,加粗序列分别与位于DEV US7和US8基因间插入位点上、下游序列同源,用于引入同源重组臂。DEV-tk-F/DEV-tk-R和JD-F/JD-R为鉴定引物对,前者用于扩增DEV基因、后者用于验证外源基因是否正确插入到BAC基因组中。1.6 pEP-pro-Es质粒的构建
采用表1引物进行PCR扩增获得的pCAG、pSV40、pRSV、pgB（MDV）和p1.8k（MDV）基因,在5′和3′端分别引入了酶切位点,将片段用对应的酶进行双酶切之后插入到pEP-BGH-Es相应的酶切位点中,从而分别用pCAG、pSV40、pRSV、pgB（MDV）和p1.8k（MDV）替换pEP-BGH-Es上的pCMV启动子,构建不同启动子调控Es基因的质粒。通过PCR和酶切鉴定筛选获得阳性克隆,送上海博尚生物公司测序,获得阳性克隆分别命名为pEP-pCAG-Es、pEP-pSV40-Es、pEP-pRSV-Es、pEP-pgB（MDV）-Es和pEP-p1.8k（MDV）- Es（总称为pEP-pro-Es）。1.7 重组鸭瘟病毒突变体BAC克隆的构建
各重组鸭瘟突变体BAC克隆通过“Red E/T两步重组”[33,34]获得。以pEP-pro-E451-dk质粒为模板,用引物对pDEV vac-in-s/pDEV vac-in-as或pDEV vac-in-s（p1.8k,pgB）/pDEV vac-in-as （序列见表1）扩增长度为3 191—4 493 bp不等的PCR片段,将PCR片段电转化至pDEV-EF1/GS1783感受态细胞,PCR片段通过自身序列上的DEV基因组同源臂将pro-Es-BGH-pA表达框和筛选基因卡那霉素（Kan）插入到pDEV-EF1。获得的重组BAC克隆用Pst I酶切鉴定,筛选到含Kan标记的重组BAC克隆中间体pDEV-pro-kan.Es。之后通过pEP-BGH-Es质粒载体上自身携带的同源序列将Kan基因去除,获得只含有pro-Es-BGH-pA表达框的重组子。抽提重组子BAC DNA,通过Pst I酶切,筛选出正确克隆（pDEV-pro- Es）。最后采用鉴定引物对DEV-tk-F/DEV-tk-R和JD-F /JD-R（序列见表1）进行PCR扩增,将扩出片段送出测序。1.8 重组病毒的拯救
采用碱裂解法抽提各个pDEV-pro-Es,依照磷酸钙转染试剂盒操作说明转染CEFs,置于37℃ 5% CO2培养箱中培养,待出现70%—80%荧光斑之后收集细胞及上清,即为拯救的病毒液,分别命名为rDEV-pCAG-Es、rDEV-pSV40-Es、rDEV-pRSV-Es、rDEV-pgB（MDV）-Es、rDEV-p1.8k（MDV）-Es（总称为rDEV-pro-Es）。1.9 重组病毒蚀斑大小的测定
将rDEV-pCAG-Es、rDEV-pSV40-Es、rDEV-pRSV- Es、rDEV-pgB（MDV）-Es、rDEV-p1.8k（MDV）-Es、rDEV-Es及rDEV-EF1病毒液稀释接种于单层CEFs上,90 min后用PBS（pH7.2）洗涤一次,铺上1.5%的甲基纤维素,置于细胞培养箱培养48 h,在荧光显微镜下将每种病毒荧光蚀斑各拍100张,用Image J软件测量病毒的蚀斑面积,并计算出平均值,以rDEV-EF1病毒株蚀斑面积为参考,将其面积设定为100%,以之为标准将其他株病毒蚀斑面积换算成百分比。用SPSS11.5软件对这些数据进行统计学分析。1.10 TUMV E蛋白DIII和DEV UL44多克隆抗体效价测定
经间接ELISA法测得兔抗TUMV E蛋白DIII结构域多克隆抗体效价和小鼠抗DEV UL44多克隆抗体效价皆为1﹕64 000。1.11 蛋白表达分析
分别接种rDEV-pCAG-Es、rDEV-pSV40-Es、rDEV-pRSV-Es、rDEV-pgB（MDV）-Es、rDEV-p1.8k （MDV）-Es和rDEV-Es及对照毒株rDEV-EF1[2]于CEFs细胞中,培养至细胞出现90%以上荧光,收集细胞培养液上清,同时用PBS（pH7.2）洗涤细胞,收获细胞沉淀。收集的培养液上清采用millipore的蛋白超滤管（30k）进行100倍浓缩,将收集的细胞及上清样品用上样缓冲液处理后,进行SDS-PAGE电泳并转移至硝酸纤维素NC膜上,转膜样品制备3份。取出NC膜,放入含10%脱脂牛奶的封闭液中4 ℃过夜。一份以兔抗TUMV E蛋白DIII结构域多克隆抗体（1﹕500稀释）和鼠源GFP单抗（1﹕1 000稀释）作为一抗,一份以小鼠抗DEV UL44多克隆抗体（1﹕500稀释）,另一份以小鼠抗beta-actin单克隆作为一抗（1﹕1 000稀释）;二抗对应的采用HRP标记的山羊抗鼠IgG（1﹕5 000稀释）和HRP标记的山羊抗兔（1﹕5 000稀释）于37℃孵育1 h,最后用Western BLoT Chemiluminescence HRP Substrate化学发光底物进行显色,并置于凝胶成像仪进行条带曝光。2 结果
2.1 重组鸭瘟病毒BAC突变体克隆的鉴定
分别提取各重组鸭瘟病毒BAC突变体克隆及Kan中间体,用Pst I酶切,电泳鉴定（图1）。结果表明DNA电泳图谱和以GenBank（登录号KF487736.1）收录的鸭瘟病毒参考序列进行预测的基本一致。电泳及测序结果也表明,以DEV-tk-F和DEV-tk-R、JD-F和JD-R引物对扩出的PCR片段（电泳图见图2）与预期一致,说明外源基因已按照预期插入鸭瘟病毒疫苗株基因组中。图1
新窗口打开|下载原图ZIP|生成PPT图1重组突变体BAC克隆的Pst I 酶切鉴定
Fig. 1Analysis of recombinant BAC mutants by Pst I digestion
1: pDEV-EF1; 2: pDEV-Es; 3: pDEV-kan. pRSV. Es; 4: pDEV-pRSV. Es; 5: pDEV-kan.pSV40. Es; 6: pDEV-pSV40. Es; 7: pDEV-kan.pCAG. Es; 8: pDEV-pCAG. Es; 9: pDEV-kan.p1.8k(MDV). Es; 10: pDEV-p1.8k (MDV). Es; 11: pDEV-kan.pgB(MDV). Es; 12: pDEV-pgB(MDV). Es
图2
新窗口打开|下载原图ZIP|生成PPT图2重组BAC突变体克隆的PCR鉴定
泳道1—6 是以DEV-tk-F和DEV-tk-R引物对扩增片段;7—12 是以JD-F和JD-R引物对扩增片段。M1：DL2000(2000,1000, 750, 500, 250, 100 bp); 1. pDEV-EF1 (553 bp); 2. pDEV-pSV40. Es (553 bp); 3. pDEV- pRSV. Es (553 bp); 4. pDEV-pCAG. Es(553 bp); 5. pDEV-p1.8k (MDV). Es (553 bp); 6. pDEV-pgB(MDV). Es (553 bp); M2: 250 bp marker(4500, 3000, 2250, 1500, 1000, 750, 500, 250 bp); 7. pDEV-EF1(641 bp); 8. pDEV-pSV40. Es(2747 bp); 9. pDEV-pRSV. Es(2745 bp); 10. pDEV- pCAG. Es(3989 bp); 11. pDEV-p1.8k(MDV). Es (2687 bp);12. pDEV-pgB (MDV). Es (3037 bp)
Fig. 2Analysis of recombinant BAC mutants by PCR amplification
2.2 重组病毒的拯救
转染细胞48 h后于荧光显微镜下观察,发现转染孔内出现荧光蚀斑,继续培养至70%—80%细胞出现荧光蚀斑之后收集细胞和上清,即为拯救重组病毒（图3）。图3
新窗口打开|下载原图ZIP|生成PPT图3拯救重组病毒
Fig. 3The rescued viruses (100×)
2.3 病毒蚀斑面积测定
病毒蚀斑面积测定表明,与rDEV-EF1相比,rDEV- Es、rDEV-pCAG-Es、rDEV-pSV40-Es、rDEV-pRSV-Es蚀斑面积分别较rDEV-EF1减少了35.26%、15.61%、10.22%、11.41%,而rDEV-p1.8k（MDV）-Es 和rDEV- pgB（MDV）-Es蚀斑面积分别较rDEV-EF1增加了21%和35.66%（图4）。图4
新窗口打开|下载原图ZIP|生成PPT图4各重组病毒在细胞上的蚀斑面积测定和比较
Fig. 4Plaque area measurement of recombinant viruses on CEFs
2.4 外源蛋白Es表达分析
将各重组毒株感染CEFs,分别收集细胞和50倍浓缩的细胞培养液上清,将细胞样品进行SDS-PAGE电泳及转膜,做3份重复,分别以兔抗TUMV E蛋白DIII结构域多克隆抗体、小鼠抗GFP单克隆抗体和小鼠抗DEV UL44多克隆抗体、小鼠抗beta-actin单克隆抗体作为一抗,进行Western blotting检测,结果表明以小鼠抗GFP单克隆抗体和小鼠抗DEV UL44多克隆抗体作为一抗检测的膜在进rDEV-Es、rDEV- pRSV-Es、rDEV-pCAG-Es感染的细胞样品在约49—52kD处显出特异性目的蛋白条带（图5-A）,在28 kD处显示出内参GFP条带,与预测基本一致,说明蛋白获得了表达。其他参考蛋白beta-actin、UL44皆在相应位置显出了条带。以beta-actin为内参,用Image J软件分析各样品表达条带灰度值比值（Es/beta-actin）,结果表明含pCMV、pCAG、pRSV所表达的蛋白强度比值分别是1.367、0.698和3.679,说明pRSV启动子活性最强,rDEV-pRSV- Es感染细胞中Es蛋白表达量较rDEV-Es提高了169.12%（图5-A）。细胞培养上清样品中明显检测到GFP的表达,但未检测到目的蛋白和病毒蛋白UL44（图5-B）。图5
新窗口打开|下载原图ZIP|生成PPT图5Western blotting方法检测重组病毒感染细胞（A）和浓缩上清（B）中Es的表达
Fig. 5Western blotting analysis of Es protein expressed in recombinant virus-infected CEFs
1: rDEV-Es; 2: rDEV-pSV40-Es; 3: rDEV-pCAG-Es; 4: rDEV-pRSV-Es; 5: rDEV-p1.8(MDV) -Es; 6: rDEV-pgB(MDV)-Es; M: EasySee Western Marker (90, 75, 60,40, 25 kD); 7: rDEV-EF1
3 讨论
感染性细菌染色体克隆（BACs）是疱疹病毒基因组操作的最主要的平台,基于该平台,国内外****广泛开展了疱疹病毒基因缺失疫苗和重组活载体疫苗的研究,目前马立克氏病毒（MDV）和伪狂犬病毒（PRV）的基因缺失疫苗和重组活载体疫苗已经获得了成功,并广泛用于实际生产中。课题组曾将鸭瘟病毒疫苗株基因组插入到BAC质粒构建了鸭瘟病毒全基因组的感染性克隆,获得了DEV疫苗株的反向遗传系统[24],尝试将以鸡为宿主进行密码子优化的DTMUV E基因插入到DEV的反向遗传系统,但外源基因E的表达水平不高[25]。之后又摸索了E基因的表达形式,包括是否前缀信号肽、是否去掉跨膜区、是否进行基因优化和以哪个宿主密码子为参考进行基因优化效果最佳都进行了摸索,筛选到了一种E蛋白表达量较高的E基因的表达形式（即去掉跨膜区、以鸭为宿主进行密码子优化的E451基因）[30]。本研究中选用了几种常用的启动子：巨细胞病毒CMV早期启动子（pCMV）、猿猴病毒SV40的早期启动子（pSV40）、Rous肉瘤病毒RSV启动子（pRSV）、复合启动子pCAG、鸡源病毒MDV 1.8k基因（p1.8k（MDV））和gB基因启动子（pgB（MDV））,其中,pCMV和pRSV调控E蛋白表达的效果最佳,究其原因,可能CMV与DEV同属疱疹病毒科;RSV天然宿主为鸡,因此更适合。本研究中几株病毒的蚀斑面积较亲本毒株或多或少都有些波动,说明启动子影响了病毒在细胞间的传播能力,但启动子的替换不影响病毒在细胞上的增殖及在细胞间的传播。
是否外源基因的表达水平与抗体生成水平有关,目前存在着一定的争议。LI等[27]、TSUKAMOTO等[35]的研究表明强启动子更有利于外源基因的表达,且外源基因的表达量与诱导机体产生的抗体水平成正相关性,高水平的抗体更利于机体对强毒株的免疫保护。但也有相反的研究结论,MA等的研究表明外源蛋白表达水平过高,会与母源抗体发生中和,从而使得外源蛋白抗体水平下降,反而导致免疫保护效果不佳[36]。综上,以DEV作为载体表达外源蛋白需要维持外源蛋白表达量到一个度,太低不足以诱导机体产生抗体,太高要么会有毒性要么会和抗体相中和。此外,本研究选用的是去除跨膜区的E基因的截短形式,最新的研究表明截短形式的E蛋白依然能保护青年鸭免受DTMUV强毒的攻击[37]。DEV和DTMUV共同的自然宿主是鸭和鹅,而本试验是在鸡源细胞上开展的,是否在其他宿主细胞或者宿主体内蛋白表达量会有差异,需要进一步研究。至于重组病毒rDEV- pRSV-Es以及其他几株病毒免疫效果如何最终需要通过后续的动物试验进行综合评价。
4 结论
本研究成功筛选获得了一种调控鸭坦布苏病毒Es在重组鸭瘟病毒载体中高效表达的启动子pRSV。同时也获得了一株高效表达鸭坦布苏病毒外源基因Es的重组鸭瘟病毒rDEV-pRSV-Es。该研究为鸭瘟病毒—坦布苏病毒二联苗的研制奠定了基础。参考文献 原文顺序
文献年度倒序
文中引用次数倒序
被引期刊影响因子
DOI:10.3390/v12010026URL [本文引用: 1]
DOI:10.1016/j.vaccine.2019.11.072URLPMID:31862196 [本文引用: 2]
Despite decades of vaccination, surveillance, and biosecurity measures, H5N2 low pathogenicity avian influenza (LPAI) virus infections continue in Mexico and neighboring countries. One explanation for tenacity of H5N2 LPAI in Mexico is the antigenic divergence of circulating field viruses compared to licensed vaccines due to antigenic drift. Our phylogenetic analysis indicates that the H5N2 LPAI viruses circulating in Mexico and neighboring countries since 1994 have undergone antigenic drift away from vaccine seed strains. Here we evaluated the efficacy of a new recombinant fowlpox virus vector containing an updated H5 insert (rFPV-H5/2016), more relevant to the current strains circulating in Mexico. We tested the vaccine efficacy against a closely related subcluster 4 Mexican H5N2 LPAI (2010 H5/LP) virus and the historic H5N2 HPAI (1995 H5/HP) virus in White Leghorn chickens. The rFPV-H5/2016 vaccine provided hemagglutinin inhibition (HI) titers pre-challenge against viral antigens from both challenge viruses in almost 100% of the immunized birds, with no differences in number of birds seroconverting or HI titers among all tested doses (1.5, 2.0, and 3.1 log10 mean tissue culture infectious doses/bird). The vaccine conferred 100% clinical protection and a significant decrease in oral and cloacal virus shedding from 1995 H5/HP virus challenged birds when compared to the sham controls at all tested doses. Virus shedding titers from vaccinated 2010 H5/LP virus challenged birds significantly decreased compared to sham birds especially at earlier time points. Our results confirm the efficacy of the new rFPV-H5/2016 against antigenic drift of LPAI virus in Mexico and suggest that this vaccine would be a good candidate, likely as a primer in a prime-boost vaccination program.
DOI:10.1007/978-1-0716-0255-3_8URLPMID:32002905 [本文引用: 1]
Free radicals of oxidative and nitrosative stress can trigger both pro-inflammatory and anti-inflammatory responses. In the transplant setting, reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced at the rejection site by different cell types including endothelial cells and macrophages. In particular, production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) seems to play an important role in promoting inflammation after exposure to inflammatory stimuli. In xenotransplantation, NO produced by iNOS upregulate multiple vasoactive substances, cytokines, chemokines, and growth factors, whereas production of NO by endothelial nitric oxide synthase (eNOS) could confer a protective effect to the graft. Accordingly, further research is needed to better understand the associated mechanisms in order to enhance protection and prevent tissue damage. Here, we describe simple methods to determine the redox state in serum that could be applied to animal models such as for xenotransplantation studies, as well as to clinical samples. Notably, caution should be taken when interpreting results of ROS and RNS measurements due to this dual role of free radicals in protecting and injuring the graft.
DOI:10.1016/j.chom.2019.10.001URLPMID:31668877 [本文引用: 1]
Maternal infection with Zika virus (ZIKV) can lead to microcephaly and other congenital abnormalities of the fetus. Although ZIKV vaccines that prevent or reduce viremia in non-pregnant mice have been described, a maternal vaccine that provides complete fetal protection would be desirable. Here, we show that adenovirus (Ad) vector-based ZIKV vaccines induce potent neutralizing antibodies that confer robust maternal and fetal protection against ZIKV challenge in pregnant, highly susceptible IFN-alphabetaR(-/-) mice. Moreover, passive transfer of maternal antibodies from vaccinated dams protected pups against post-natal ZIKV challenge. These data suggest that Ad-based ZIKV vaccines may be able to provide protection in pregnant females against fetal ZIKV transmission in utero as well as in infants against ZIKV infection after birth.
[本文引用: 1]
DOI:10.3390/v11090784URLPMID:31450681 [本文引用: 1]
Newcastle disease (ND) is responsible for significant economic losses in the poultry industry. The disease is caused by virulent strains of Avian avulavirus 1 (AAvV-1), a species within the family Paramyxoviridae. We developed a recombinant construct based on the herpesvirus of turkeys (HVT) as a vector expressing two genes: F and HN (HVT-NDV-F-HN) derived from the AAvV-1 genotype VI (
DOI:10.1016/j.virusres.2019.197648URLPMID:31279828 [本文引用: 1]
Throughout the past few decades, numerous viral species have been generated as vaccine vectors. Every viral vector has its own distinct characteristics. For example, the family herpesviridae encompasses several viruses that have medical and veterinary importance. Attenuated herpesviruses are developed as vectors to convey heterologous immunogens targeting several serious and crucial pathogens. Some of these vectors have already been licensed for use in the veterinary field. One of their prominent features is their capability to accommodate large amount of foreign DNA, and to stimulate both cell-mediated and humoral immune responses. A better understanding of vector-host interaction builds up a robust foundation for the future development of herpesviruses-based vectors. At the time, many molecular tools are applied to enable the generation of herpesvirus-based recombinant vaccine vectors such as BAC technology, homologous and two-step en passant mutagenesis, codon optimization, and the CRISPR/Cas9 system. This review article highlights the most important techniques applied in constructing recombinant herpesviruses vectors, advantages and disadvantages of each recombinant herpesvirus vector, and the most recent research regarding their use to control major animal diseases.
DOI:10.1016/j.vaccine.2009.10.089URL [本文引用: 1]
Abstract
Recently developed viral-vectored HIV vaccine candidates, despite achieving high levels transgene expression and inducing high magnitude immune responses to HIV, have faced limitations related to anti-vector immunity. In contrast, lentiviral vectors (LV) have been shown to be less sensitive to anti-vector neutralizing activity, while displaying desirable characteristics, such as transduction of non-dividing cells, including antigen-presenting cells, and long-term transgene expression.We have developed VRX1023, an HIV-based LV expressing HIV Gag, Pol and Rev under the control of the native HIV LTR. In mice, this vector induced significant mucosal and systemic cellular and humoral responses against HIV after sub-cutaneous injection. Similarly to other viral vectors, this LV candidate can be effectively used in DNA prime, LV boost strategies, where it elicited as high as 21% HIV Gag-specific CD8 responses as measured by intracellular cytokine staining. Moreover, anti-vector immunity is not an obstacle to repeated LV administrations, as shown by improved anti-HIV responses compared to single LV immunization. In head to head comparisons with Ad5 vectors expressing the same vaccine payload, VRX1023 elicited higher and more persistent cellular and antibody responses to HIV than its adenoviral counterpart. In preparation for clinical use, manufacturing scale-up of a highly purified VRX1023 vector lot following cGMP was successfully achieved without altering the robust immunogenicity observed with the research-grade vector.
VRX1023, in addition to competing favorably with existing vectors such as Ad5 for anti-HIV immune responses, demonstrates unique features likely to address some of the pitfalls of current vector-based HIV vaccine strategies.
DOI:10.1186/s12977-018-0449-7URLPMID:30285769 [本文引用: 1]
Vectored gene delivery of HIV-1 broadly neutralizing antibodies (bNAbs) using recombinant adeno-associated virus (rAAV) is a promising alternative to conventional vaccines for preventing new HIV-1 infections and for therapeutically suppressing established HIV-1 infections. Passive infusion of single bNAbs has already shown promise in initial clinical trials to temporarily decrease HIV-1 load in viremic patients, and to delay viral rebound from latent reservoirs in suppressed patients during analytical treatment interruptions of antiretroviral therapy. Long-term, continuous, systemic expression of such bNAbs could be achieved with a single injection of rAAV encoding antibody genes into muscle tissue, which would bypass the challenges of eliciting such bNAbs through traditional vaccination in naive patients, and of life-long repeated passive transfers of such biologics for therapy. rAAV delivery of single bNAbs has already demonstrated protection from repeated HIV-1 vaginal challenge in humanized mouse models, and phase I clinical trials of this approach are underway. Selection of which individual, or combination of, bNAbs to deliver to counter pre-existing resistance and the rise of escape mutations in the virus remains a challenge, and such choices may differ depending on use of this technology for prevention versus therapy.
URL [本文引用: 1]
【Objective】 A strain of replication deficient recombinant adenovirus encoding HA of H3N2 subtype swine influenza virus (SIV) was generated and the immune efficacy was evaluated in mice. 【Method】 To construct a recombinant adenovirus shuttle plasmid pDC315-H3HA-EGFP, HA gene of A/Swine/Guangdong/9/2005(H3N2) amplified by PCR from the recombinant plasmid pMD18-HA was sub-cloned into pIRES2-EGFP. The gene fragment containing HA and EGFP was then inserted into adenovirus shuttle plasmid pDC315. A replication-defective recombinant adenovirus expressing HA gene (rAd-H3HA-EGFP) was generated by cotransfecting the recombinant shuttle plasmid pDC315-HA-EGFP and the backbone plasmid pBHGlox△E1, E3Cre in HEK293 cells. The recombinant adenovirus was screened by the typical cytopathic effect and expression of EGFP gene in HEK293 cells. The immunogenicity of the recombinant adenovirus was evaluated by inoculating 6-week-old BALB/c mice, through detecting specific antibody titer and protection against the virus challenge. 【Result】 HA gene was recombined into the genome of the recombinant adenovirus rAd-H3HA-EGFP, and HA protein could be efficiently expressed in vitro. The TCID50 of the rAd-H3HA-EGFP was evaluated as 1.58×1010•mL-1 after propagation and purification. Mice inoculated with 108 TCID50 were protected against the challenge with H3 subtype SIV and accompanied with high titer of antibodies. 【Conclusion】 A strain of replication-defective adenovirus rAd-H3HA-EGFP with good immunogenicity was constructed, which would lay a foundation for the development of the engineered live virus vectored vaccine of H3 subtype of SIV.
URL [本文引用: 1]
【Objective】 A strain of replication deficient recombinant adenovirus encoding HA of H3N2 subtype swine influenza virus (SIV) was generated and the immune efficacy was evaluated in mice. 【Method】 To construct a recombinant adenovirus shuttle plasmid pDC315-H3HA-EGFP, HA gene of A/Swine/Guangdong/9/2005(H3N2) amplified by PCR from the recombinant plasmid pMD18-HA was sub-cloned into pIRES2-EGFP. The gene fragment containing HA and EGFP was then inserted into adenovirus shuttle plasmid pDC315. A replication-defective recombinant adenovirus expressing HA gene (rAd-H3HA-EGFP) was generated by cotransfecting the recombinant shuttle plasmid pDC315-HA-EGFP and the backbone plasmid pBHGlox△E1, E3Cre in HEK293 cells. The recombinant adenovirus was screened by the typical cytopathic effect and expression of EGFP gene in HEK293 cells. The immunogenicity of the recombinant adenovirus was evaluated by inoculating 6-week-old BALB/c mice, through detecting specific antibody titer and protection against the virus challenge. 【Result】 HA gene was recombined into the genome of the recombinant adenovirus rAd-H3HA-EGFP, and HA protein could be efficiently expressed in vitro. The TCID50 of the rAd-H3HA-EGFP was evaluated as 1.58×1010•mL-1 after propagation and purification. Mice inoculated with 108 TCID50 were protected against the challenge with H3 subtype SIV and accompanied with high titer of antibodies. 【Conclusion】 A strain of replication-defective adenovirus rAd-H3HA-EGFP with good immunogenicity was constructed, which would lay a foundation for the development of the engineered live virus vectored vaccine of H3 subtype of SIV.
URL [本文引用: 1]
以国家品种资源库编目入库的云南地方稻种资源 61 2 1份为材料 ,以 31个分类、形态及产量性状为基本数据研究了云南地方稻种资源的核心种质取样方案。取样方案包括分组原则、组内取样比例的确定和组内取样方法 ,分组原则为按丁颖分类体系、程王分类体系、云南稻作生态区、行政地区和单一性状分组及不分组的大随机 ;组内取样比例的确定有平方根法、对数法、遗传多样性法和简单比例法 ;组内取样采用随机法和聚类法。结果表明 ,遗传多样性指数、表型方差、表型频率方差、变异系数和表型保留比例等 5个参数可作为检验各取样方案所得的核心种质的指标 ,通过检验可知 ,以丁颖和程王两种分类体系为分组原则、以平方根或对数法确定组内取样比例采用聚类法在组内进行取样是比较好的取样方案
URL [本文引用: 1]
以国家品种资源库编目入库的云南地方稻种资源 61 2 1份为材料 ,以 31个分类、形态及产量性状为基本数据研究了云南地方稻种资源的核心种质取样方案。取样方案包括分组原则、组内取样比例的确定和组内取样方法 ,分组原则为按丁颖分类体系、程王分类体系、云南稻作生态区、行政地区和单一性状分组及不分组的大随机 ;组内取样比例的确定有平方根法、对数法、遗传多样性法和简单比例法 ;组内取样采用随机法和聚类法。结果表明 ,遗传多样性指数、表型方差、表型频率方差、变异系数和表型保留比例等 5个参数可作为检验各取样方案所得的核心种质的指标 ,通过检验可知 ,以丁颖和程王两种分类体系为分组原则、以平方根或对数法确定组内取样比例采用聚类法在组内进行取样是比较好的取样方案
DOI:10.1016/j.vaccine.2019.08.026URLPMID:31471151 [本文引用: 1]
Ducks play a key role in the maintenance and spread of avian influenza viruses (AIVs) in nature, and control of AIVs in ducks has important implications for AIV eradication from poultry. We previously constructed a recombinant duck enteritis virus (DEV), rDEVus78HA, that expresses the HA gene of an H5N1 AIV and showed that rDEVus78HA immunization provides complete protection against both DEV and H5N1 AIV challenge in specific-pathogen-free ducks. In this study, we performed a 60-week clinical trial and found that this rDEVus78HA vaccine can function as a bivalent vaccine in farmed ducks against lethal challenge with DEV and H5N1 virus. Moreover, we found that rDEVus78HA-vaccinated ducks were efficiently protected against challenges with recently isolated heterologous H5N6 and H5N8 viruses. Our results demonstrate that rDEVus78HA could be extremely valuable for the control of DEV and H5 AIVs in ducks.
DOI:10.1016/j.vetmic.2019.04.022URLPMID:31030839 [本文引用: 1]
Newcastle disease virus (NDV) is a major threat to poultry worldwide. Virulent Newcastle disease virus infection can cause 100% morbidity and mortality in chickens. Vaccination is the most effective way to prevent and control NDV outbreaks in poultry. Previously, we demonstrated that a duck enteritis virus (DEV) vaccine strain is a promising vector to generate recombinant vaccines in chickens. Here, we constructed two recombinant DEVs expressing the F protein (rDEV-F) or HN protein (rDEV-HN) of NDV. We then evaluated the protective efficacy of these recombinant DEVs in specific-pathogen-free chickens. rDEV-F induced 100% protection of chickens from lethal NDV challenge after a single dose of 10(4) TCID50, whereas rDEV-HN did not induce effective protection. rDEV-F may therefore serve as a promising vaccine candidate for chickens. This is the first report of a DEV-vectored vaccine providing robust protection against lethal NDV infection in chickens.
DOI:10.3390/v10020081URLPMID:29438322 [本文引用: 1]
Duck-targeted vaccines to protect against avian influenza are critically needed to aid in influenza disease control efforts in regions where ducks are endemic for highly pathogenic avian influenza (HPAI). Duck enteritis virus (DEV) is a promising candidate viral vector for development of vaccines targeting ducks, owing to its large genome and narrow host range. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system is a versatile gene-editing tool that has proven beneficial for gene modification and construction of recombinant DNA viral vectored vaccines. Currently, there are two commonly used methods for gene insertion: non-homologous end-joining (NHEJ) and homology-directed repair (HDR). Owing to its advantages in efficiency and independence from molecular requirements of the homologous arms, we utilized NHEJ-dependent CRISPR/Cas9 to insert the influenza hemagglutinin (HA) antigen expression cassette into the DEV genome. The insert was initially tagged with reporter green fluorescence protein (GFP), and a Cre-Lox system was later used to remove the GFP gene insert. Furthermore, a universal donor plasmid system was established by introducing double bait sequences that were independent of the viral genome. In summary, we provide proof of principle for generating recombinant DEV viral vectored vaccines against the influenza virus using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.
DOI:10.1038/s41598-017-01554-1URLPMID:28469192 [本文引用: 2]
Duck enteritis virus (DEV), duck tembusu virus (DTMUV), and highly pathogenic avian influenza virus (HPAIV) H5N1 are the most important viral pathogens in ducks, as they cause significant economic losses in the duck industry. Development of a novel vaccine simultaneously effective against these three viruses is the most economical method for reducing losses. In the present study, by utilizing a clustered regularly interspaced short palindromic repeats (CRISPR)/associated 9 (Cas9)-mediated gene editing strategy, we efficiently generated DEV recombinants (C-KCE-HA/PrM-E) that simultaneously encode the hemagglutinin (HA) gene of HPAIV H5N1 and pre-membrane proteins (PrM), as well as the envelope glycoprotein (E) gene of DTMUV, and its potential as a trivalent vaccine was also evaluated. Ducks immunized with C-KCE-HA/PrM-E enhanced both humoral and cell-mediated immune responses to H5N1 and DTMUV. Importantly, a single-dose of C-KCE-HA/PrM-E conferred solid protection against virulent H5N1, DTMUV, and DEV challenges. In conclusion, these results demonstrated for the first time that the CRISPR/Cas9 system can be applied for modification of the DEV genome rapidly and efficiently, and that recombinant C-KCE-HA/PrM-E can serve as a potential candidate trivalent vaccine to prevent H5N1, DTMUV, and DEV infections in ducks.
DOI:10.3389/fmicb.2016.01613URLPMID:27777571 [本文引用: 1]
As causative agents of duck viral hepatitis, duck hepatitis A virus type 1 (DHAV-1) and type 3 (DHAV-3) causes significant economic losses in the duck industry. However, a licensed commercial vaccine that simultaneously controls both pathogens is currently unavailable. Here, we generated duck enteritis virus recombinants (rC-KCE-2VP1) containing both VP1 from DHAV-1 (VP1/DHAV-1) and VP1 from DHAV-3 (VP1/DHAV-3) between UL27 and UL26. A self-cleaving 2A-element of FMDV was inserted between the two different types of VP1, allowing production of both proteins from a single open reading frame. Immunofluorescence and Western blot analysis results demonstrated that both VP1 proteins were robustly expressed in rC-KCE-2VP1-infected chicken embryo fibroblasts. Ducks that received a single dose of rC-KCE-2VP1 showed potent humoral and cellular immune responses and were completely protected against challenges of both pathogenic DHAV-1 and DHAV-3 strains. The protection was rapid, achieved as early as 3 days after vaccination. Moreover, viral replication was fully blocked in vaccinated ducks as early as 1 week post-vaccination. These results demonstrated, for the first time, that recombinant rC-KCE-2VP1 is potential fast-acting vaccine against DHAV-1 and DHAV-3.
DOI:10.1007/s00705-016-3077-3URLPMID:27709401 [本文引用: 1]
H9 subtype avian influenza viruses (AIVs) remain a significant burden in the poultry industry and are considered to be one of the most likely causes of any new influenza pandemic in humans. As ducks play an important role in the maintenance of H9 viruses in nature, successful control of the spread of H9 AIVs in ducks will have significant beneficial effects on public health. Duck enteritis virus (DEV) may be a promising candidate viral vector for aquatic poultry vaccination. In this study, we constructed a recombinant DEV, rDEV-UL2-HA, inserting the hemagglutinin (HA) gene from duck-origin H9N2 AIV into the UL2 gene by homologous recombination. One-step growth analyses showed that the HA gene insertion had no effect on viral replication and suggested that the UL2 gene was nonessential for virus growth in vitro. In vivo tests further showed that the insertion of the HA gene in place of the UL2 gene did not affect the immunogenicity of the virus. Moreover, a single dose of 10(3) TCID50 of rDEV-UL2-HA induced solid protection against lethal DEV challenge and completely prevented H9N2 AIV viral shedding. To our knowledge, this is the first report of a DEV-vectored vaccine providing robust protection against both DEV and H9N2 AIV virus infections in ducks.
DOI:10.1016/j.antiviral.2016.03.003URLPMID:26946113 [本文引用: 1]
To design an alternative vaccine for control of infectious bronchitis in chickens, three recombinant duck enteritis viruses (rDEVs) expressing the N, S, or S1 protein of infectious bronchitis virus (IBV) were constructed using conventional homologous recombination methods, and were designated as rDEV-N, rDEV-S, and rDEV-S1, respectively. Chickens were divided into five vaccinated groups, which were each immunized with one of the rDEVs, covalent vaccination with rDEV-N & rDEV-S, or covalent vaccination with rDEV-N & rDEV-S1, and a control group. An antibody response against IBV was detectable and the ratio of CD4(+)/CD8(+) T-lymphocytes decreased at 7 days post-vaccination in each vaccinated group, suggesting that humoral and cellular responses were elicited in each group as early as 7 days post-immunization. After challenge with a homologous virulent IBV strain at 21 days post-immunization, vaccinated groups showed significant differences in the percentage of birds with clinical signs, as compared to the control group (p < 0.01), as the two covalent-vaccination groups and the rDEV-S group provided better protection than the rDEV-N- or rDEV-S1-vaccinated group. There was less viral shedding in the rDEV-N & rDEV-S- (2/10) and rDEV-N & rDEV-S1- (2/10) vaccinated groups than the other three vaccinated groups. Based on the clinical signs, viral shedding, and mortality rates, rDEV-N & rDEV-S1 covalent vaccination conferred better protection than use of any of the single rDEVs.
DOI:10.1186/s12985-015-0354-9URLPMID:26263920 [本文引用: 1]
BACKGROUND: Highly pathogenic avian influenza virus (AIV) subtype H5N1 remains a threat to poultry. Duck enteritis virus (DEV)-vectored vaccines expressing AIV H5N1 hemagglutinin (HA) may be viable AIV and DEV vaccine candidates. METHODS: To facilitate the generation and further improvement of DEV-vectored HA(H5) vaccines, we first constructed an infectious clone of DEV Chinese vaccine strain C-KCE (DEV(C-KCE)). Then, we generated a DEV-vectored HA(H5) vaccine (DEV-H5(UL55)) based on the bacterial artificial chromosome (BAC) by inserting a synthesized HA(H5) expression cassette with a pMCMV IE promoter and a consensus HA sequence into the noncoding area between UL55 and LORF11. The immunogenicity and protective efficacy of the resulting recombinant vaccine against DEV and AIV H5N1 were evaluated in both ducks and chickens. RESULTS: The successful construction of DEV BAC and DEV-H5(UL55) was verified by restriction fragment length polymorphism analysis. Recovered virus from the BAC or mutants showed similar growth kinetics to their parental viruses. The robust expression of HA in chicken embryo fibroblasts infected with the DEV-vectored vaccine was confirmed by indirect immunofluorescence and western blotting analyses. A single dose of 10(6) TCID50 DEV-vectored vaccine provided 100 % protection against duck viral enteritis in ducks, and the hemagglutination inhibition (HI) antibody titer of AIV H5N1 with a peak of 8.2 log2 was detected in 3-week-old layer chickens. In contrast, only very weak HI titers were observed in ducks immunized with 10(7) TCID50 DEV-vectored vaccine. A mortality rate of 60 % (6/10) was observed in 1-week-old specific pathogen free chickens inoculated with 10(6) TCID50 DEV-vectored vaccine. CONCLUSIONS: We demonstrate the following in this study. (i) The constructed BAC is a whole genome clone of DEV(C-KCE). (ii) The insertion of an HA expression cassette sequence into the noncoding area between UL55 and LORF11 of DEV(C-KCE) affects neither the growth kinetics of the virus nor its protection against DEV. (iii) DEV-H5(UL55) can generate a strong humoral immune response in 3-week-old chickens, despite the virulence of this virus observed in 1-week-old chickens. (iv) DEV-H5(UL55) induces a weak HI titer in ducks. An increase in the HI titers induced by DEV-vectored HA(H5) will be required prior to its wide application.
DOI:10.1186/s13567-015-0174-3URLPMID:25889564 [本文引用: 1]
Duck is susceptible to many pathogens, such as duck hepatitis virus, duck enteritis virus (DEV), duck tembusu virus, H5N1 highly pathogenic avian influenza virus (HPAIV) in particular. With the significant role of duck in the evolution of H5N1 HPAIV, control and eradication of H5N1 HPAIV in duck through vaccine immunization is considered an effective method in minimizing the threat of a pandemic outbreak. Consequently, a practical strategy to construct a vaccine against these pathogens should be determined. In this study, the DEV was examined as a candidate vaccine vector to deliver the hemagglutinin (HA) gene of H5N1, and its potential as a polyvalent vaccine was evaluated. A modified mini-F vector was inserted into the gB and UL26 gene junction of the attenuated DEV vaccine strain C-KCE genome to generate an infectious bacterial artificial chromosome (BAC) of C-KCE (vBAC-C-KCE). The HA gene of A/duck/Hubei/xn/2007 (H5N1) was inserted into the C-KCE genome via the mating-assisted genetically integrated cloning (MAGIC) to generate the recombinant vector pBAC-C-KCE-HA. A bivalent vaccine C-KCE-HA was developed by eliminating the BAC backbone. Ducks immunized with C-KCE-HA induced both the cross-reactive antibodies and T cell response against H5. Moreover, C-KCE-HA-immunized ducks provided rapid and long-lasting protection against homologous and heterologous HPAIV H5N1 and DEV clinical signs, death, and primary viral replication. In conclusion, our BAC-C-KCE is a promising platform for developing a polyvalent live attenuated vaccine.
DOI:10.1016/j.virusres.2011.04.013URL [本文引用: 1]
We report on the generation of an infectious bacterial artificial chromosome (BAC) clone of duck enteritis virus (DEV) and a vectored DEV vaccine expressing hemagglutinin (H5) of high pathogenicity H5N1 avian influenza virus (AIV). For generation of the DEV BAC, we inserted mini-F vector sequences by homologous recombination in lieu of the UL44 (gC) gene of DEV isolate 2085. DNA of the resulting in recombinant virus v2085-GFP Delta gC was electroporated into Escherichia coli and a full-length DEV BAC clone (p2085) was recovered. Transfection of p2085 into chicken embryo cells resulted in DEV-specific plaques exhibiting green autofluorescence. A gC-negative mutant, v2085 Delta gC, was generated by deleting mini-F vector sequences by using Cre-Lox recombination, and a revertant virus v2085 Delta gC-R was constructed by co-transfection of p2085 with UL44 sequences. Finally, AIV H5 was inserted into p2085, and high-level H5 expression of the v2085_H5 virus was detected by indirect immunofluorescence and western blotting. Plaque area measurements showed that v2085 Delta gC plaques were significantly increased (12%) over those of parental 2085 virus or the v2085 Delta gC-R revertant virus (ANOVA, P<0.05), while plaque areas of the H5- or GFP-expressing DEV mutants were significantly smaller. There was no significant difference between DEV with respect to virus titers determined after trypsinization titration of infected cells, while virus titers of infected-cell supernatants revealed significant reductions in case of the gC-negative viruses of more than 700-fold when compared to parental 2085 or v2085 Delta gC-R. Cell-associated virus titers of gC-negative DEV also showed significant reduction of 50-500-fold (ANOVA, P < 0.05). We conclude that (i) absence of DEV gC results in increased plaque sizes in vitro, (ii) gC plays a role in DEV egress, and (iii) generation of an infectious DEV clone allows rapid generation of vectored vaccines. (C) 2011 Elsevier B.V.
DOI:10.1128/JVI.05420-11URL [本文引用: 1]
Ducks play an important role in the maintenance of highly pathogenic H5N1 avian influenza viruses (AIVs) in nature, and the successful control of AIVs in ducks has important implications for the eradication of the disease in poultry and its prevention in humans. The inactivated influenza vaccine is expensive, labor-intensive, and usually needs 2 to 3 weeks to induce protective immunity in ducks. Live attenuated duck enteritis virus (DEV; a herpesvirus) vaccine is used routinely to control lethal DEV infections in many duck-producing areas. Here, we first established a system to generate the DEV vaccine strain by using the transfection of overlapping fosmid DNAs. Using this system, we constructed two recombinant viruses, rDEV-ul41HA and rDEV-us78HA, in which the hemagglutinin (HA) gene of the H5N1 virus A/duck/Anhui/1/06 was inserted and stably maintained within the ul41 gene or between the us7 and us8 genes of the DEV genome. Duck studies indicated that rDEV-us78HA had protective efficacy similar to that of the live DEV vaccine against lethal DEV challenge; importantly, a single dose of 10(6) PFU of rDEV-us78HA induced complete protection against a lethal H5N1 virus challenge in as little as 3 days postvaccination. The protective efficacy against both lethal DEV and H5N1 challenge provided by rDEV-ul41HA inoculation in ducks was slightly weaker than that provided by rDEV-us78HA. These results demonstrate, for the first time, that recombinant DEV is suitable for use as a bivalent live attenuated vaccine, providing rapid protection against both DEV and H5N1 virus infection in ducks.
DOI:10.1016/j.vaccine.2013.10.035URLPMID:24144474 [本文引用: 1]
The duck enteritis virus (DEV) may be a promising candidate viral vector for an aquatic poultry vaccination that can protect against multiple pathogens because it has a very large genome and a narrow host range. Recently, we described two DEV recombinants that contained deletions of the viral US2 or gIgE genes. The hemagglutinin (HA) gene of an H5N1-type highly pathogenic avian influenza virus (HPAIV) of goose origin was inserted into the deletion sites to construct two rDEVs expressing the AIV HA antigen. The resulting rDEV-DeltagIgE-HA or rDEV-DeltaUS2-HA recombinant DEV viruses were used to infect duck embryo fibroblasts. Reverse transcription PCR, immunofluorescence and western blot analysis results indicated that rDEV-DeltagIgE-HA and rDEV-DeltaUS2-HA were successfully expressed in duck embryo fibroblasts (DEFs). To investigate whether the HA gene could be stably maintained in the recombinant viruses, the viruses were passaged in DEFs 18 times. The HA gene in both recombinants could be detected by PCR amplification. The immunized four-week-old ducks induced specific antibodies against DEV and AIV HA and were protected against challenge infections with DEV AV1221 viruses.
DOI:10.1016/j.vaccine.2014.07.082URL [本文引用: 3]
A newly emerged tembusu virus that causes egg-drop has been affecting ducks in China since 2010. Currently, no vaccine is available for this disease. A live attenuated duck enteritis virus (DEV; a herpesvirus) vaccine has been used routinely to control lethal DEV in ducks since the 1960s. Here, we constructed two recombinant DEVs by transfecting overlapping fosmid DNAs. One virus, rDEV-TE, expresses the truncated form of the envelope glycoprotein (TE) of duck tembusu virus (DTMUV), and the other virus, rDEV-PrM/TE, expresses both the TE and pre-membrane proteins (PrM). Animal study demonstrated that both recombinant viruses induced measurable anti-DTMUV neutralizing antibodies in ducks. After two doses of recombinant virus, rDEV-PrM/TE completely protected ducks from DTMUV challenge, whereas rDEV-TE only conferred partial protection. These results demonstrate that recombinant DEV expressing the TE and pre-membrane proteins is protective and can serve as a potential candidate vaccine to prevent DTMUV infection in ducks. (C) 2014 Elsevier Ltd.
DOI:10.3390/v6062428URLPMID:24956180 [本文引用: 3]
Duck Tembusu virus (DTMUV) is a recently emerging pathogenic flavivirus that has resulted in a huge economic loss in the duck industry. However, no vaccine is currently available to control this pathogen. Consequently, a practical strategy to construct a vaccine against this pathogen should be determined. In this study, duck enteritis virus (DEV) was examined as a candidate vaccine vector to deliver the envelope (E) of DTMUV. A modified mini-F vector was inserted into the SORF3 and US2 gene junctions of the attenuated DEV vaccine strain C-KCE genome to generate an infectious bacterial artificial chromosome (BAC) of C-KCE (vBAC-C-KCE). The envelope (E) gene of DTMUV was inserted into the C-KCE genome through the mating-assisted genetically integrated cloning (MAGIC) strategy, resulting in the recombinant vector, pBAC-C-KCE-E. A bivalent vaccine C-KCE-E was generated by eliminating the BAC backbone. Immunofluorescence and western blot analysis results indicated that the E proteins were vigorously expressed in C-KCE-E-infected chicken embryo fibroblasts (CEFs). Duck experiments demonstrated that the insertion of the E gene did not alter the protective efficacy of C-KCE. Moreover, C-KCE-E-immunized ducks induced neutralization antibodies against DTMUV. These results demonstrated, for the first time, that recombinant C-KCE-E can serve as a potential bivalent vaccine against DEV and DTMUV.
DOI:10.3864/j.issn.0578-1752.2016.14.015URL [本文引用: 1]
【目的】鸭瘟和小鹅瘟是番鸭和鹅的两种重要传染病,鸭瘟最主要的防治措施是定期接种鸭瘟病毒减毒活疫苗。根据2012年国际病毒分类委员会（ICTV）的报告,DEV被归为疱疹病毒科的α疱疹病毒亚科马立克氏病毒属。疱疹病毒如伪狂犬病毒、马立克氏病毒、火鸡疱疹病毒等已广泛用于病毒活载体的研究,而近几年也有关于鸭瘟病毒（DEV）作为疫苗活载体的报道。为了为免疫防控鸭瘟和小鹅瘟提供新手段,本研究拟在鸭瘟病毒疫苗株感染性克隆的基础上,构建表达小鹅瘟病毒（GPV）主要免疫原蛋白VP2的重组病毒rDEV-VP2,并研究其生物学特性,进而探讨重组病毒rDEV-VP2作为防治DEV和GPV的二联重组活载体疫苗的可能性。【方法】将密码子优化的GPV VP2基因通过常规基因克隆的方法插入转移载体pEP-BGH-end,构建含有GPV VP2表达框pCMV-VP2-BGH-pA的重组表达质粒。在鸭瘟病毒（DEV）疫苗株细菌人工染色体克隆pDEV-EF1的基础上,通过“Red E/T”两步重组法将GPV VP2基因表达框插入到DEV US7和US8基因之间构建了突变体克隆pDEV-VP2。利用磷酸钙法转染鸡胚成纤维细胞（CEFs）拯救获得重组病毒rDEV-VP2和删除Bac质粒序列的rDEV-VP2-Cre,并对重组病毒细胞体外生长曲线、蚀斑大小和VP2蛋白表达情况进行测定。将rDEV-VP2接种番鸭,在不同时间采集血清,采用间接ELISA法检测血清中GPV VP2抗体产生情况。【结果】间接免疫荧光检测和Western blotting分析表明,外源蛋白VP2在CEFs细胞成功表达。病毒生长曲线和蚀斑大小测定结果显示,rDEV-VP2在CEFs细胞上的增殖滴度与亲本株相比无显著差异,表明外源基因VP2的插入不影响rDEV重组病毒的增殖。动物试验结果表明,7日龄雏番鸭接种rDEV-VP2可以诱导产生针对GPV VP2的抗体,免疫后3周抗体阳性率为50%（4/8）。【结论】本实验将小鹅瘟病毒的主要免疫原基因VP2插入到DEV疫苗株基因组的US7和US8基因间构建了表达该免疫原性基因的重组鸭瘟病毒细菌人工染色体,继而在鸡胚成纤维细胞（CEFs）上拯救获得了重组病毒rDEV-VP2,病毒细胞生长特性与亲本株基本一致,且能诱导鸭体产生GPV VP2特异性的抗体。该研究为研制DEV-GPV二联重组活载体疫苗奠定了基础。
DOI:10.3864/j.issn.0578-1752.2016.14.015URL [本文引用: 1]
【目的】鸭瘟和小鹅瘟是番鸭和鹅的两种重要传染病,鸭瘟最主要的防治措施是定期接种鸭瘟病毒减毒活疫苗。根据2012年国际病毒分类委员会（ICTV）的报告,DEV被归为疱疹病毒科的α疱疹病毒亚科马立克氏病毒属。疱疹病毒如伪狂犬病毒、马立克氏病毒、火鸡疱疹病毒等已广泛用于病毒活载体的研究,而近几年也有关于鸭瘟病毒（DEV）作为疫苗活载体的报道。为了为免疫防控鸭瘟和小鹅瘟提供新手段,本研究拟在鸭瘟病毒疫苗株感染性克隆的基础上,构建表达小鹅瘟病毒（GPV）主要免疫原蛋白VP2的重组病毒rDEV-VP2,并研究其生物学特性,进而探讨重组病毒rDEV-VP2作为防治DEV和GPV的二联重组活载体疫苗的可能性。【方法】将密码子优化的GPV VP2基因通过常规基因克隆的方法插入转移载体pEP-BGH-end,构建含有GPV VP2表达框pCMV-VP2-BGH-pA的重组表达质粒。在鸭瘟病毒（DEV）疫苗株细菌人工染色体克隆pDEV-EF1的基础上,通过“Red E/T”两步重组法将GPV VP2基因表达框插入到DEV US7和US8基因之间构建了突变体克隆pDEV-VP2。利用磷酸钙法转染鸡胚成纤维细胞（CEFs）拯救获得重组病毒rDEV-VP2和删除Bac质粒序列的rDEV-VP2-Cre,并对重组病毒细胞体外生长曲线、蚀斑大小和VP2蛋白表达情况进行测定。将rDEV-VP2接种番鸭,在不同时间采集血清,采用间接ELISA法检测血清中GPV VP2抗体产生情况。【结果】间接免疫荧光检测和Western blotting分析表明,外源蛋白VP2在CEFs细胞成功表达。病毒生长曲线和蚀斑大小测定结果显示,rDEV-VP2在CEFs细胞上的增殖滴度与亲本株相比无显著差异,表明外源基因VP2的插入不影响rDEV重组病毒的增殖。动物试验结果表明,7日龄雏番鸭接种rDEV-VP2可以诱导产生针对GPV VP2的抗体,免疫后3周抗体阳性率为50%（4/8）。【结论】本实验将小鹅瘟病毒的主要免疫原基因VP2插入到DEV疫苗株基因组的US7和US8基因间构建了表达该免疫原性基因的重组鸭瘟病毒细菌人工染色体,继而在鸡胚成纤维细胞（CEFs）上拯救获得了重组病毒rDEV-VP2,病毒细胞生长特性与亲本株基本一致,且能诱导鸭体产生GPV VP2特异性的抗体。该研究为研制DEV-GPV二联重组活载体疫苗奠定了基础。
DOI:10.1016/j.vaccine.2016.10.008URLPMID:27742216 [本文引用: 2]
The vaccine efficacy of recombinant viruses can be influenced by many factors. Accordingly, the activity of promoters has been one of the major factors affecting the antigen expression and protection rate. In the present study, two recombinant Marek's disease virus type 1 (MDV1) vaccines containing the VP2 gene of infectious bursal disease virus (IBDV) under control of different promoters were generated from overlapping fosmid DNAs. The rMDV-Pec-VP2 virus containing the VP2 gene under control of the Pec promoter (CMV enhancer and chicken beta-actin chimera promoter) demonstrated higher VP2 expression and stronger antibody response against IBDV in chickens than the rMDV-CMV-VP2 virus using the CMV promoter. After IBDV lethal challenge in specific-pathogen-free chickens, rMDV-Pec-VP2 provided complete protection against developing mortality, clinical signs, and the formation of bursal lesions, which was better than that provided by rMDV-CMV-VP2. Our findings indicate that the protective efficacy of the recombinant MDV1 vaccine against IBDV highly correlates with VP2 expression. This recombinant MDV1 vaccine expressing VP2 could have significant potential as a bivalent vaccine against both virulent IBDV and MDV infections in chickens.
DOI:10.1186/1743-422X-10-328URLPMID:24195756 [本文引用: 1]
BACKGROUND: Duck enteritis virus (DEV) is the causative agent of duck viral enteritis, which causes an acute, contagious and lethal disease of many species of waterfowl within the order Anseriformes. In recent years, two laboratories have reported on the successful construction of DEV infectious clones in viral vectors to express exogenous genes. The clones obtained were either created with deletion of viral genes and based on highly virulent strains or were constructed using a traditional overlapping fosmid DNA system. Here, we report the construction of a full-length infectious clone of DEV vaccine strain that was cloned into a bacterial artificial chromosome (BAC). METHODS: A mini-F vector as a BAC that allows the maintenance of large circular DNA in E. coli was introduced into the intergenic region between UL15B and UL18 of a DEV vaccine strain by homologous recombination in chicken embryoblasts (CEFs). Then, the full-length DEV clone pDEV-vac was obtained by electroporating circular viral replication intermediates containing the mini-F sequence into E. coli DH10B and identified by enzyme digestion and sequencing. The infectivity of the pDEV-vac was validated by DEV reconstitution from CEFs transfected with pDEV-vac. The reconstructed virus without mini-F vector sequence was also rescued by co-transfecting the Cre recombinase expression plasmid pCAGGS-NLS/Cre and pDEV-vac into CEF cultures. Finally, the in vitro growth properties and immunoprotection capacity in ducks of the reconstructed viruses were also determined and compared with the parental virus. RESULTS: The full genome of the DEV vaccine strain was successfully cloned into the BAC, and this BAC clone was infectious. The in vitro growth properties of these reconstructions were very similar to parental DEV, and ducks immunized with these viruses acquired protection against virulent DEV challenge. CONCLUSIONS: DEV vaccine virus was cloned as an infectious bacterial artificial chromosome maintaining full-length genome without any deletions or destruction of the viral coding sequence, and the viruses rescued from the DEV-BAC clone exhibited wild-type phenotypes both in vitro and in vivo. The generated infectious clone will greatly facilitate studies on the individual genes of DEV and applications in gene deletion or live vector vaccines.
URL [本文引用: 1]
摘要：为开展鸭瘟病毒-鸭坦布苏病毒二联苗的研究，将密码子优化的鸭坦布苏病毒（DTMUV）E基因插入转移载体pEP\|BGH\|end构建了pEP\|BGH\|E重组表达质粒。在鸭瘟病毒疫苗株细菌人工染色体（pDEV\|vac）的基础上，首先通过Red E/T两步重组法构建了EF1启动子替换GFP基因CMV启动子的pDEV\|EF1突变体克隆，并将pEP\|BGH\|E质粒上的重组表达框pCMV\|E\|BGH\|pA再次通过两步重组克隆至pDEV\|EF1突变体的US7和US8基因之间，构建了携带有外源基因DTMUV E的突变体克隆pDEV\|E。用磷酸钙法转染鸡胚成纤维细胞（CEFs）获得了重组病毒rDEV\|E。病毒蚀斑大小测定结果显示，rDEV\|E在CEFs上的蚀斑面积较亲本株相比稍有减少。Western blotting分析表明，外源蛋白E在病毒感染的CEFs细胞中成功表达。
URL [本文引用: 1]
摘要：为开展鸭瘟病毒-鸭坦布苏病毒二联苗的研究，将密码子优化的鸭坦布苏病毒（DTMUV）E基因插入转移载体pEP\|BGH\|end构建了pEP\|BGH\|E重组表达质粒。在鸭瘟病毒疫苗株细菌人工染色体（pDEV\|vac）的基础上，首先通过Red E/T两步重组法构建了EF1启动子替换GFP基因CMV启动子的pDEV\|EF1突变体克隆，并将pEP\|BGH\|E质粒上的重组表达框pCMV\|E\|BGH\|pA再次通过两步重组克隆至pDEV\|EF1突变体的US7和US8基因之间，构建了携带有外源基因DTMUV E的突变体克隆pDEV\|E。用磷酸钙法转染鸡胚成纤维细胞（CEFs）获得了重组病毒rDEV\|E。病毒蚀斑大小测定结果显示，rDEV\|E在CEFs上的蚀斑面积较亲本株相比稍有减少。Western blotting分析表明，外源蛋白E在病毒感染的CEFs细胞中成功表达。
DOI:10.1007/s12033-019-00206-1URLPMID:31482466 [本文引用: 2]
In our previous study, a recombinant duck enteritis virus (DEV) delivering codon-optimized E gene (named as E-ch) of duck Tembusu virus (DTMUV) optimized referring to chicken's codon bias has been obtained based on the infectious bacterial artificial chromosome (BAC) clone of duck enteritis virus vaccine strain pDEV-EF1, but the expression level of E-ch in recombinant virus rDEV-E-ch-infected cells was very low. To optimize DTMUV E gene expression delivered by the vectored DEV, different forms of E gene (collectively called EG) including origin E gene (E-ori), truncated E451-ori gene, codon-optimized E-dk gene optimized referring to duck's codon bias, as well as the truncated E451-ch and E451-dk, Etpa-ori and Etpa-451-ori, which contain prefixing chick TPA signal peptide genes, were cloned into transfer vector pEP-BGH-end, and several recombinant plasmids pEP-BGH-EG were constructed. Then the expression cassettes pCMV-EG-polyABGH amplified from pEP-BGH-EG by PCR were inserted into US7/US8 gene intergenic region of pDEV-EF1 by two-step Red/ET recombination, 7 strain recombinant mutated BAC clones pDEV-EG carrying different E genes were constructed. Next, the recombinant viruses rDEV-EG were reconstituted from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. Western blot analysis showed that E or E451 protein is expressed in rDEV-E-ori, rDEV-E-ch, rDEV-Etpa-ori, rDEV-E451-ori, rDEV-E451-dk, and rDEV-E451-ch-infected CEFs, and protein expression level in rDEV-E451-dk-infected CEFs is the highest. These studies have laid a foundation for developing bivalent vaccine controlling DEV and DTMUV infection.
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DOI:10.2144/000112096URLPMID:16526409 [本文引用: 1]
Red recombination using PCR-amplified selectable markers is a well-established technique for mutagenesis of large DNA molecules in Escherichia coli. The system has limited efficacy and versatility, however, for markerless modifications including point mutations, deletions, and particularly insertions of longer sequences. Here we describe a procedure that combines Red recombination and cleavage with the homing endonuclease I-SceI to allow highly efficient, PCR-based DNA engineering without retention of unwanted foreign sequences. We applied the method to modification of bacterial artificial chromosome (BAC) constructs harboring an infectious herpesvirus clone to demonstrate the potential of the mutagenesis technique, which was used for the insertion of long sequences such as coding regions or promoters, introduction of point mutations, scarless deletions, and insertion of short sequences such as an epitope tag. The system proved to be highly reliable and efficient and can be adapted for a variety of different modifications of BAC clones, which are fundamental tools for applications as diverse as the generation of transgenic animals and the construction of gene therapy or vaccine vectors.
Jeff Braman(ed.),
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DOI:10.1128/jvi.76.11.5637-5645.2002URLPMID:11991992 [本文引用: 1]
Marek's disease herpesvirus is a vaccine vector of great promise for chickens; however, complete protection against foreign infectious diseases has not been achieved. In this study, two herpesvirus of turkey recombinants (rHVTs) expressing large amounts of infectious bursal disease virus (IBDV) VP2 antigen under the control of a human cytomegalovirus (CMV) promoter or CMV/beta-actin chimera promoter (Pec promoter) (rHVT-cmvVP2 and rHVT-pecVP2) were constructed. rHVT-pecVP2, which expressed the VP2 antigen approximately four times more than did rHVT-cmvVP2 in vitro, induced complete protection against a lethal IBDV challenge in chickens, whereas rHVT-cmvVP2 induced 58% protection. All of the chickens vaccinated with rHVT-pecVP2 had a protective level of antibodies to the VP2 antigen at the time of challenge, whereas only 42 and 67% of chickens vaccinated with rHVT-cmvVP2 or the conventional live IBDV vaccine, respectively, had the antibodies. The antibody level of chickens vaccinated with rHVT-pecVP2 increased for 16 weeks, and the peak antibody level persisted throughout the experiment. The serum antibody titer at 30 weeks of age was about 20 or 65 times higher than that of chickens vaccinated with rHVT-cmvVP2 or the conventional live vaccine, respectively. rHVT-pecVP2, isolated consistently for 30 weeks from the vaccinated chickens, expressed the VP2 antigen after cultivation, and neither nucleotide mutations nor deletion in the VP2 gene was found. These results demonstrate that the amount of VP2 antigen expressed in the HVT vector was correlated with the vaccine efficacy against lethal IBDV challenge, and complete protective immunity that is likely to persist for the life of the chickens was induced.
DOI:10.1016/j.jviromet.2014.05.023URL [本文引用: 1]
On the basis of recent studies, much attention has been given to recombinant MDV (rMDV)-based vaccines. During the construction of rMDV, the activity of promoters to transcribe foreign genes is one of the major factors that can affect protective efficacy. To investigate the transcription activity and efficacy of five different promoters, the advantage of an existing rMDV BAC infectious clone that had been previously constructed was used to construct rMDVs. The expression cassette of the hemagglutinin gene (HA) from a low pathogenic avian influenza virus (LPAIV) H9N2 strain was inserted into the US2 region under five selected promoters. These five promoters included three MDV endogenous promoters (the promoter for the gB gene and a bi-directional promoter in both directions for pp38 (ppp38) and 1.8 kb RNA transcripts (p1.8 kb)), and two exogenous promoters (CMV and SV40). Among these five promoters, the CMV promoter demonstrated the highest activity, followed by p1.8 kb and SV40, which had a similar transcriptional activity level. Two of the MDV endogenous promoters showed much lower transcriptional activities, particularly the promoter ppp38, which had the lowest activity. The results of the in vivo experiment proved that none of the three recombinant viruses of rGX-CMV-HA, rGX-SV40-HA and rGX-p1.8kb-HA provided protection in SPF chickens. Chickens vaccinated with rGX-pPP38-HA induced 50% and rGX-gB-HA induced 25% protection against the challenge with H9N2, respectively. (C). 2014 Elsevier B.V.
DOI:10.1016/j.vetmic.2019.108508URLPMID:31902493 [本文引用: 1]
Duck Tembusu virus (DTMUV) is a major pathogen of duck industry in China. In the current study, we generated different constructs containing envelope (E) protein, pre-membrane-envelope (prM-E) protein, and C-terminally truncated E protein of the DTMUV. The constructed proteins could induce specific antibody responses in young ducks. When ducklings were immunized with the constructed proteins, they were 100% protected against DTMUV infection. Furthermore, the fluorescent signal of the truncated E protein was stronger than other constructed proteins, when Bac-to-Bac baculovirus expression system was applied. Our data demonstrated that the truncated E protein used in the current study could be applied as a potential vaccine candidate to control DTMUV infection in young ducks.
