,1, 于康英1, 张婷2, 陆睿2, 潘生强1, 成勇,2, 周鸣鸣,1Preparation and Expression of rhPA/GH Double Transgenic Rabbits
SONG ShaoZheng,1, YU KangYing1, ZHANG Ting2, LU Rui2, PAN ShengQiang1, CHENG Yong,2, ZHOU MingMing,1通讯作者:
责任编辑: 林鉴非
收稿日期:2019-07-14接受日期:2020-11-25网络出版日期:2021-01-16
| 基金资助: |
Received:2019-07-14Accepted:2020-11-25Online:2021-01-16
作者简介 About authors
宋绍征,E-mail：
摘要
关键词：
Abstract
Keywords：
PDF (1082KB)元数据多维度评价相关文章导出EndNote|Ris|Bibtex收藏本文
本文引用格式
宋绍征, 于康英, 张婷, 陆睿, 潘生强, 成勇, 周鸣鸣. rhPA/GH双转基因兔的制备及rhPA表达检测[J]. 中国农业科学, 2021, 54(2): 412-421 doi:10.3864/j.issn.0578-1752.2021.02.016
SONG ShaoZheng, YU KangYing, ZHANG Ting, LU Rui, PAN ShengQiang, CHENG Yong, ZHOU MingMing.
开放科学（资源服务）标识码（OSID）：
0 引言
【研究意义】血栓病是一种严重威胁人类生命健康的常见多发病,溶栓疗法是目前临床上应用最广泛而有效的一种治疗方法[1,2,3]。人组织纤溶酶原激活剂（tissue-type plasminogen activator, tPA）是由血管内皮细胞合成并分泌的一种丝氨酸蛋白酶,能够高效特异地溶解血栓,属于一种良好的第二代溶栓药物[4]。重组人纤溶酶原激活剂（recombinant human plasminogen activator, rhPA）是天然tPA的重组突变体,属于新型第三代溶栓药物,具有较天然tPA更加优越的溶栓功效[5]。随着医学的不断发展,高产优质的新型溶栓药物日趋重要。因此,研究如何稳定提高rhPA的表达水平和产量,对于开发新型溶栓药物具有重要的指导意义,也为今后其它重组医药蛋白的高效表达研究和工业化生产奠定了基础。【前人研究进展】转基因技术可应用于动物遗传育种、生物反应器、疾病模型和器官移植等领域。目前,外源目的基因的表达沉默是转基因动物研究遇到的一个重要瓶颈,虽然利用友好位点（Rosa26、Hipp11、Pifs501）、定点整合（ZFNs、TALENs、CRISPR/Cas9）、优化顺式作用元件（启动子、内含子、增强子）等措施可克服或减轻基因表达沉默现象,但仍然存在一定的局限性[6,7]。因此,在优化提高转基因生物目的基因表达水平方面需要有针对性地灵活选择应用合理的技术手段。有研究证明,通过双基因共整合获得的转基因生物能够产生协同促进作用,提高目的基因的表达水平[8,9]。例如王林楠等[10]通过慢病毒介导NEP1-40和NT-3双基因转染大鼠神经干细胞,结果显示NEP1-40和NT-3 mRNA相对表达量及蛋白表达量均明显地高于单基因转染。陈宁等[11]将NGF与BMP2双基因转染大鼠BMSCs,显示双基因转染组的目的蛋白表达量明显地提高。徐莉等[12]利用双启动子构建人补体调节蛋白DAF和MCP转染小鼠成纤维细胞NIH3T3,DAF和MCP双基因均能够协同高效共表达,且表达水平明显得到提高。周慧[13]和吕本浩[14]等其他研究者也得出了双基因转染的目的蛋白表达水平高于单基因这一结论。【本研究切入点】家兔是一种应用最广泛的实验动物之一,与大型动物牛羊相比,具有排卵多、妊娠期短、繁殖力强、全年多发情等优点;与小鼠相比,具有泌乳量高、适合于生产重组医药蛋白等优点,可以填补大型和小型动物间的“空白”[15]。生长激素（growth hormone, GH）是一种由垂体前叶分泌的类似催乳素结构的蛋白质,控制着β-casein与α-LA受体的激活,产生协同作用,具有促进乳腺生长发育及维持泌乳的功能[16,17],这预示着将GH转入动物体内可能会在一定程度上提高目的基因的表达水平。因此,在转基因动物遗传育种方面发挥着重要的作用。但是,将rhPA和GH双基因共整合入转基因兔体内以期提高rhPA表达量的相关研究较少见报道,GH是否能够协同促进目的基因rhPA在转基因兔乳腺中高效表达值得进一步研究。【拟解决的关键问题】本研究以rhPA单转基因兔[15]（PCL25/rhPA乳腺特异性表达载体,以β-casein作为调控序列）为试验兔,其超排获得的受精卵通过显微注射的方法额外转入GH,旨在提高转基因兔乳腺表达rhPA的含量,为将来制备高表达rhPA转基因动物和遗传育种提供了新思路、新方法,也为其他重组医药蛋白的生产奠定了基础。1 材料与方法
试验于2018—2019年在扬州大学江苏省转基因动物制药工程研究中心实验室和无锡太湖学院基础医学实验室完成。1.1 材料
1.1.1 质粒与菌种 PCL25/rhPA（tPA 的L、F、E、K1区缺失突变体）和PCL25/GH质粒、菌种保存于实验室,是以山羊β-casein为调控元件、CMV为启动子的哺乳动物乳腺特异性表达载体（图1）,已在山羊细胞和兔个体上进行表达验证[15,18]。图1
新窗口打开|下载原图ZIP|生成PPT图1乳腺特异性表达载体PCL25/rhPA与PCL25/GH构建图及PCR检测原理图
Fig. 1Constructions and PCR detection schematic diagram of PCL25/rhPA and PCL25/GH mammary gland specific expression vector
1.1.2 引物PCR 引物设计借助于Primer Premier 5.0 软件完成,引物由上海生工生物工程技术有限公司合成（表1）。
Table 1
表1
表1PCR扩增引物序列
Table 1
| 引物名称 Primer name | 引物序列 Primer sequence (5°-3°) |
|---|---|
| gGH-F | CCGCTCGAGCGGATGATGGCTGCAGGCCCCCGGA |
| gGH-R | CCGCTCGAGCGGCTAGAAGGCACAGCTGGCCTCCCCG |
| CMV/tPA-F | CGTGGATAGCGGTTTGA |
| CMV/tPA-R | GAGCCCTCCTTTGATGC |
新窗口打开|下载CSV
1.1.3 主要试剂 FSH（宁波三生药业）,HCG（丽珠制药厂）,速眠新II（军需大学兽医研究所）,FBS（HyClone）,透明质酸酶（Sigma）,Zoletil50（Virbac）,蛋白酶K（Sigma）,M2（Sigma）,M16（Sigma）,鼠抗tPA单克隆抗体（Santa Cruz）,羊抗鼠单克隆抗体IgG-HRP（Santa Cruz）;DNA 胶纯化回收试剂盒购自QIAGEN 公司;各种限制性内切酶和DNA聚合酶购自宝生物工程（大连）有限公司;其他未说明试剂均为国产分析纯,分别购自上海药剂,上海生工生物工程有限公司,南京生兴生物有限公司。
1.1.4 实验动物 rhPA单转基因兔（品种为新西兰兔,标号K06、K10、K17,以山羊β-casein为调控元件,且已验证表达）和正常非转基因新西兰兔,均单笼饲养于江苏省转基因动物制药工程研究中心清洁级兔房,温度20℃,光照12 h（7:00—19:00）,颗粒饲料,自由饮水,环境良好。
1.2 方法
1.2.1 显微注射用基因片段的准备 PCL25/GH质粒通过NotⅠ/ SalⅠ双酶切而线性化,1%琼脂糖凝胶电泳分离不同大小分子量的基因片段,去除原核基因片段,使用QIAGEN DNA胶纯化回收试剂盒回收真核基因片段供显微注射用。使用TE缓冲液（5 mmol·L-1 Tris,pH 7.4 0.1 mmol·L-1 EDTA）溶解稀释至 5 ng·μL-1,-20℃保存。1.2.2 兔超数排卵与同期发情 挑取未发情的rhPA单转基因兔（K06、K10、K17）作为供体,后肢肌肉注射FSH,每次10 IU/只,早晚各一次（间隔12 h）,连续3 d。第4天上午7:00肌肉注射FSH 5 IU/只,晚上19:00耳缘静脉注射hCG 100 IU/只,人工辅助与正常新西兰公兔配种后再合笼。第5天中午12:00无菌手术取卵[18]。在供体兔配种的同时,挑取8—10月龄自然发情的成年健康新西兰母兔作为受体,耳缘静脉注射hCG 100 IU/只,以备次日手术移植。母兔发情的主要标志为阴道黏膜呈潮红色、分泌粘液较多。
1.2.3 双转基因兔的制备 供体兔注射hCG后17 h,麻醉（皮下注射阿托品1 mg·kg -1,15 min后耳缘静脉注射zoletil-50 7.5 mg·kg-1）,仰卧保定,无菌手术输卵管冲卵,回收受精卵,在体视显微镜下观察、计数。在荧光倒置显微镜（IX70,Olympus）下,将显微注射基因片段导入受精卵的原核内,置于38 ℃、5% CO2、饱和湿度的培养箱中培养 30 min后,手术移植到同步发情的受体母兔输卵管内,待孕。
1.2.4 转基因兔的整合筛选 无菌剪取新生仔兔的耳尖组织约2 mm3,添加含200 μg蛋白酶K的组织裂解液,55℃消化过夜,苯酚/氯仿抽提法提取基因组,-20℃预冷的无水乙醇沉淀DNA,进行PCR检测。针对rhPA和GH两种基因,分别设计了两对引物（表1）,检测位点示意图如图1所示,其中CMV/tPA引物用于rhPA转基因检测,PCR 参数为：95℃ 预变性 5 min ;94℃变性 45 s,55℃退火 45 s,72℃延伸 1 min,共30个循环;72℃延伸10 min。gGH引物用于GH转基因检测,PCR 参数为：95℃ 预变性 5 min ;94℃变性 45 s,58℃退火30 s,72℃延伸 45 s,共30个循环;72℃延伸10 min。PCR反应产物进行1%琼脂糖凝胶电泳,确定条带大小是否正确。
1.2.5 ELISA 表达检测 转基因母兔配种怀孕,分娩后挤奶,收集乳汁。乳汁离心,10 000×g,30 min,去除上层脂肪及下层浑浊,吸取乳清,PBS稀释100倍用于检测。96-孔酶标板中每孔添加100 μL乳清和100 μL包被液（1.696 g·L-1 Na2CO3, 2.856 g·L-1 NaHCO3, pH 9.6）,4℃过夜。弃去包被液,使用含0.05% Tween-20的PBS洗涤3次,拍干。每孔加入200 μL封闭液（含10%胎牛血清的PBS）,37℃水浴2 h。使用鼠抗tPA单克隆抗体作为一抗（sc-59721, Santa Cruz）、羊抗鼠单克隆抗体IgG-HRP作为二抗（sc-2005, Santa Cruz）,分别37℃水浴2 h。洗涤后,每孔加入50 μL的显色液（5 mg OPD, 15 μL30% H2O2, 28.4 g·L-1 Na2HPO4, 19.2 g·L-1柠檬酸）,37℃避光孵育20 min,显色后酶标仪测定OD450值,并以阿替普酶（alteplase）作为标准品,绘制标准曲线,计算rhPA表达量,比较rhPA/GH双转基因兔和rhPA单转基因兔的表达水平。
1.2.6 Western blotting检测 按照常规方法对PBS稀释100倍的转基因兔乳清进行12% SDS聚丙烯酰胺凝胶电泳（SDS-PAGE）[18] 。使用转移缓冲液（1.93 g·L-1 tris, 9 g·L-1 glycine）将丙烯酰胺凝胶转移至PVDF膜,250 mA,转印3.5 h。超纯水冲洗后,37℃封闭（20 mmol·L-1 Tris, 137 mmol·L-1 NaCl, 0.1 % Tween-20, 10% fetal bovine serum,pH 7.6）,2 h。加入一抗稀释液（1﹕2 000稀释,鼠抗tPA单克隆抗体,sc-59721,Santa Cruz）,37℃孵育2 h。TTBS（20 mmol·L-1 Tris, 137 mmol·L-1 NaCl,1 % Tween-20, pH 7.6）洗涤3次后,加入二抗-HRP稀释液（1﹕2 000稀释,羊抗鼠单克隆抗体IgG-HRP,sc-2005,Santa Cruz）中,37℃孵育2 h。取出PVDF膜,PBS洗净后,添加显色液（DAB 50 mg, 0.05 mol·L-1 TB100mL, 30 μL 30% H2O2, pH7.6）,室温15 min,晾干后拍照、记录并保存。
1.2.7 双转基因兔的生长发育监测 在相同的断奶时间和饲养条件下,分别对不同生长发育阶段的rhPA/GH双转基因兔和正常非转基因兔的体重进行测量,从出生开始连续测量至6个月龄,以时间（月）为横坐标、体重（g）为纵坐标,绘制生长曲线,比较rhPA/GH双转基因兔与正常非转基因兔的生长发育情况。
2 结果
2.1 显微注射用基因片段的纯化回收
经NotⅠ/ SalⅠ双酶切质粒 PCL25/GH,使用胶纯化回收试剂盒回收后的基因片段电泳图谱见图2所示,从图中可见一约16 700 bp 大小的明亮条带,与目标显微注射基因片段的大小相同。电泳结果表明,成功地酶切和回收了大小约16 700 bp显微注射用的基因片段。图2
新窗口打开|下载原图ZIP|生成PPT图2NotⅠ/SalⅠ双酶切质粒 PCL25/GH的电泳图
P：PCL25/GH质粒双酶切;M：λ-EcoT14 DNA Marker.
Fig. 2Electrophoresis maps of plasmid PCL25/GH digested by NotⅠand SalⅠdouble enzymes
P: Plasmid PCL25/GH double digestion; M: λ-EcoT14 DNA Marker
2.2 受精卵的显微注射
正常兔受精卵为卵圆形,透明带较厚且外围一厚层粘蛋白,判断受精的标志是能够看见两个相互靠近的原核（胞质凹陷部分）,雌原核一般小于雄原核,如图3-A所示,在卵的中间部位可见两个相邻的明显凹圆,即雌雄原核。将显微注射用基因片段导入原核的操作如图3-B所示,可见注射的原核瞬间膨胀变大。图3
新窗口打开|下载原图ZIP|生成PPT图3兔受精卵原核显微注射
Fig. 3Pronuclear microinjection of rabbit fertilized eggs
2.3 PCR整合检测
通过对3只rhPA单转基因供体兔（标号K06、K10、K17）的超数排卵获得122枚卵,其中有103枚受精卵,受精率为84.4%（103/122）。挑选较好的94枚受精卵进行显微注射,经培养30 min后再挑选其中形态较好的81枚卵移植到6只同步发情的母兔输卵管中,有5只怀孕并顺利分娩,妊娠率为83.3%（5/6）,妊娠到期共出生32只仔兔。经PCR检测,共获得19只携带rhPA的转基因兔,其中11只兔（7♂,4♀）整合rhPA/GH双基因,通过PCR检测分别扩增出561 bp（rhPA）和670 bp（GH）大小条带（图4、图5）,双基因整合率为34.4%（11/32）。其中,4只rhPA/GH双转基因母兔来源亲代分别为K06供体兔2只（标号K06-1、K06-2）、K10供体兔1只（标号K10-1）、K17供体兔1只（标号K17-1）,详见表2。Table 2
表2
表2单、双转基因兔乳腺表达rhPA含量情况统计表
Table 2
| 样本Samples | K06 | K10 | K17 | K06-1 | K06-2 | K10-1 | K17-1 |
|---|---|---|---|---|---|---|---|
| rhPA表达水平 rhPA expression levels (μg·mL-1) | 42.2 | 42.8 | 15.2 | 432 | 444 | 636 | 248 |
| rhPA表达相对倍数（双基因/单基因） Expressing relative multiple of rhPA (Double-transgenic / Single-transgenic) | - | - | - | 10.2 (432/42.2) | 10.5 (444/42.2) | 14.9 (636/42.8) | 16.3 (248/15.2) |
新窗口打开|下载CSV
图4
新窗口打开|下载原图ZIP|生成PPT图4rhPA的PCR整合检测结果
1,4：正常非转基因兔（阴性对照）;2：K06-1转基因兔;3：K06-2转基因兔;5：K10-1转基因兔;6：K17-1转基因兔;M：DL2000 Marker;7：PCL25/rhPA质粒（阳性对照）
Fig. 4Detection of rhPA by PCR
1,4: Normal non-transgenic rabbit (negative control); 2:K06-1 transgenic rabbit; 3:K06-2 transgenic rabbit; 5:K10-1 transgenic rabbit; 6:K17-1 transgenic rabbit; M:DL2000 Marker; 7:PCL25/rhPA plasmid (positive control)
图5
新窗口打开|下载原图ZIP|生成PPT图5GH的PCR整合检测结果
1：正常非转基因兔（阴性对照）;2：K06-1转基因兔;3：K06-2转基因兔;4：K10-1转基因兔;5：K17-1转基因兔;6：rhPA单基因整合兔（K06）;7：PCL25/GH质粒（阳性对照）;M：DL2000 Marker
Fig. 5Detection of GH by PCR
1: Normal non-transgenic rabbit (negative control); 2: K06-1 transgenic rabbit; 3: K06-2 transgenic rabbit; 4: K10-1 transgenic rabbit; 5: K17-1 transgenic rabbit; 6: rhPA monogenic integrated rabbit (K06); 7: PCL25/GH plasmid (positive control); M: DL2000 Marker
2.4 ELISA检测
转基因兔乳清经ELISA检测表达水平的结果如图6和表2所示,其中K06兔乳清中rhPA表达量为42.2 μg·mL-1,K06-1和K06-2兔乳清中rhPA表达量分别为432、444 μg·mL-1,表达水平分别提高了约10.2倍（432/42.2）和10.5倍（444/42.2）;K10兔乳清中rhPA表达量为42.8 μg·mL-1,K10-1兔乳清中rhPA表达量为636 μg·mL-1,表达水平提高了约14.9倍（636/ 42.8）;K17兔乳清中rhPA表达量为15.2 μg·mL-1,K17-1兔乳清中rhPA表达量为248 μg·mL-1,表达水平提高了约16.3倍（248/15.2）。3只rhPA单转基因兔表达rhPA水平为15.2—42.8 μg·mL-1,而rhPA/GH双转基因兔表达rhPA水平为248—636 μg·mL-1,表达水平提高了10.2—16.3倍左右。ELISA检测结果表明,rhPA/GH双转基因兔乳腺表达rhPA水平明显高于rhPA单转基因兔,GH可协同促进rhPA在转基因兔乳腺中的高效表达。图6
新窗口打开|下载原图ZIP|生成PPT图6转基因兔乳腺表达 rhPA 浓度标准曲线图
阿替普酶浓度分别是0、0.125、0.25、0.5、1.0、2.0、4.0、8.0 μg·mL-1。所有兔乳清均使用PBS稀释100倍
Fig. 6Standard curve of rhPA expression in mammary glands of transgenic rabbits
The concentration of ateplase were 0, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0 and 8.0 μg·mL-1, respectively. All rabbits whey were diluted 100 times with PBS
2.5 Western blotting检测
单、双转基因兔乳清的Western blotting检测结果如图7所示,可见一大小约39.0 kD的条带,与阳性对照的条带大小相同。结果表明,该转基因兔乳清中成功表达的蛋白为目标产物rhPA,且其蛋白分子量大小正确,与目标蛋白一致。图7
新窗口打开|下载原图ZIP|生成PPT图7单、双转基因兔乳清的Western blotting检测
所有兔乳清均使用PBS稀释100倍
Fig. 7Western blotting detection results of single and double transgenic rabbits whey
All rabbits whey were diluted 100 times with PBS
2.6 rhPA/GH双转基因兔的生长发育情况
对整合rhPA/GH双基因的转基因兔连续测量体重6个月（表3）,并与正常非转基因兔的体重进行比较,未见明显的差异存在。其中,K10-1转基因兔前3个月体重偏轻（明显低于正常兔体重）,是由于该兔在出生的第20天生病所致,恢复健康之后,后期体重增长也恢复至正常。从兔的生长曲线（图8）上可以看出,4只整合GH的转基因兔（K06-1、K06-2、K10-1、K17-1）与2只未整合GH的正常非转基因兔（N1、N2）相比,在生长发育的不同阶段未见明显的体重差异,成长至6个月的体重均在4.0—5.0 kg之间。结果表明,GH的转入未影响兔的正常生长发育,rhPA/GH双转基因兔能够存活、正常生长发育至成年。Table 3
表3
表3正常兔和rhPA/GH双转基因兔不同生长阶段的体重测量
Table 3
| 月龄Month | N1 | N2 | K06-1 | K06-2 | K10-1 | K17-1 |
|---|---|---|---|---|---|---|
| 新出生Newborn | 53 | 66 | 61 | 58 | 63 | 49 |
| 1 | 582 | 715 | 684 | 670 | 492 | 567 |
| 2 | 1416 | 1528 | 1432 | 1587 | 921 | 1384 |
| 3 | 2587 | 3199 | 2876 | 3022 | 2165 | 2455 |
| 4 | 3621 | 4342 | 4042 | 4111 | 3758 | 3469 |
| 5 | 4125 | 4752 | 4635 | 4425 | 4225 | 4021 |
| 6 | 4367 | 4967 | 4711 | 4698 | 4412 | 4232 |
新窗口打开|下载CSV
图8
新窗口打开|下载原图ZIP|生成PPT图8转基因兔生长曲线图
N1和N2是正常非转基因兔,K06-1、K06-2、K10-1、K17-1是rhPA/GH双转基因兔
Fig. 8Growth curve of transgenic rabbits
N1 and N2 were normal non-transgenic rabbits, K06-1, K06-2, K10-1 and K17-1 were rhPA/GH double-transgenic rabbits
3 讨论
据世界卫生组织（WHO）统计分析,全球每年由于心血管病死亡的人数约为1 300 万,其中血栓类疾病占半数以上,且呈现明显的上升趋势[5,19]。目前,临床上主要采用阿替普酶（tPA）、瑞替普酶、孟替普酶、兰替普酶、替尼普酶等溶栓药物治疗血栓病。本研究的重组人纤溶酶原激活剂（rhPA）是一种新开发的第三代重组溶栓药物,具有高效、安全、特异、副作用小等优点,临床使用的溶栓药物大多是通过原核生物或哺乳动物细胞表达来生产,存在产量低、价格高等局限性,大众使用推广一直受到限制[18,19,20]。因此,如何高效、便捷地低成本生产rhPA一直是科学研究的热点。自20世纪90年代,WRIGHT等[21]在羊乳腺中成功地表达了人α-抗胰蛋白酶以来,乳腺生物反应器显示出诱人的前景,为生产重组溶栓药物提供了较大的可能性。但是rhPA或tPA在动物乳腺中的表达水平一直偏低[22]。因此,积极探索提高非乳蛋白rhPA在动物乳腺中的表达水平尤为重要。目前,提高动物转基因表达效率的方法较多[6],双转基因生物体内的两个基因能够产生协同促进作用,调控生物体基因网络系统,使外源目的基因表达水平提高[23],这是一种良好的提高目的基因表达的策略,为提高转基因兔乳腺中rhPA的表达量提供了新思路。自1920年,EVANS首次证实垂体中具有促生长的物质为生长激素以来,****们对GH进行了广泛而深入的研究,并取得了重要成果[24]。有报道证明生长激素（GH）能够与β-casein的HRE序列结合,促进受体激活,协同提高乳蛋白的特异性表达 [17,25]。虽然近年来,已有关于双基因提高外源目的基因表达水平的研究报道,例如韩操等[26]对bFGF和BMP-2双基因、王林楠等[10]对NEP1-40和NT-3双基因、陈宁等[11]对NGF与BMP2双基因的研究结果显示,双基因导入生物体内能够明显地提高目的基因的表达水平。但是,关于rhPA和GH双基因整合兔的相关研究报道较少见。
家兔是转基因试验中常用的模式生物,也是胚胎工程和乳腺生物反应器研究中应用最广泛的实验动物之一。本研究尝试利用转基因动物进行二次转基因,选择以rhPA单转基因兔（以山羊β-casein作为调控序列,且已验证表达）作为供体兔[15],通过FSH/hCG进行超数排卵获取103枚受精卵,通过原核显微注射GH,分别移植到同步发情的新西兰受体母兔体内,顺利分娩获得32只仔兔。经PCR整合检测获得11只rhPA/GH双转基因兔（7♂,4♀）,双基因整合率达到34.4%,这与目前国内外报道的转基因兔整合效率相一致[15,27-29]。对其中4只双转基因母兔（K06-1、K06-2、K10-1、K17-1）的乳腺表达水平检测结果显示,rhPA/GH双转基因兔表达目的蛋白rhPA的含量为248—636 μg·mL-1,远远高于rhPA单转基因兔（K06、K10、K17）的表达水平（15.2—42.8 μg·mL-1）。这一结果证明了GH的导入,能够大大地促进转基因兔乳腺中rhPA基因表达水平的提高。
此外,很多GH转基因动物的研究报道集中于生长激素能够调节机体生长,从而获得个体大小超越一般野生型的“超级”物种 [30]。但是,本研究中获得的GH/rhPA双转基因兔通过与正常非转基因兔的生长发育情况比较,发现GH并没有对转基因兔的生长发育产生影响,整合GH的转基因兔能够正常地生长发育至成年。一般情况下,新西兰成年兔的体重为4.0—5.0 kg[31],本试验的4只双转基因兔成长至6个月的体重均在4.0—5.0 kg之间,与正常非转基因兔的体重增长没有明显的差异。分析原因可能是试验选择的GH来源于山羊,不能够产生与在山羊体内类似的生理学作用,也不会对兔的生长发育造成影响。而且,基因表达是涉及到整合位点、表观遗传、外源基因拷贝数、相关激素水平及基因网络等多方面的影响[32],因此,相关研究仍需继续进行。
4 结论
通过二次转基因成功制备的rhPA/GH双转基因兔,不仅使双基因整合率得到了保证,也使rhPA表达水平更具有比较性。通过对兔乳清中表达rhPA含量和不同生长发育阶段的体重监测,证明了rhPA/GH双转基因兔能够明显地提高目的基因rhPA的表达量,同时GH对兔的生长发育没有造成明显的影响,这为将来制备高表达转基因兔及其它动物奠定了基础,也为转基因动物乳腺生物反应器和转基因育种建立提供了新技术、新方法。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:10.1016/S1474-4422(18)30323-5URLPMID:30507392 [本文引用: 1]
Although recent technical advances in thrombectomy have revolutionised acute stroke treatment, prevalence of disability and death related to stroke remain high. Therefore, plasminogen activators-eukaryotic, bacterial, or engineered forms that can promote fibrinolysis by converting plasminogen into active plasmin and facilitate clot breakdown-are still commonly used in the acute treatment of ischaemic stroke. Hence, plasminogen activators have become a crucial area for clinical investigation for their ability to recanalise occluded arteries in ischaemic stroke and to accelerate haematoma clearance in haemorrhagic stroke. However, inconsistent results, insufficient evidence of efficacy, or reports of side-effects in trial settings might reduce the use of plasminogen activators in clinical practice. Additionally, the mechanism of action for plasminogen activators could extend beyond the vessel lumen and involve plasminogen-independent processes, which would suggest that plasminogen activators have also non-fibrinolytic roles. Understanding the complex mechanisms of action of plasminogen activators can guide future directions for therapeutic interventions in patients with stroke.
URLPMID:29555402 [本文引用: 1]
URLPMID:27857733 [本文引用: 1]
DOI:10.1161/STROKEAHA.118.024703URLPMID:30852962 [本文引用: 1]
Background and Purpose- Telemedicine is increasingly utilized for intravenous tPA (tissue-type plasminogen activator) delivery. The comparative safety of leaving tPA-treated patients at a presenting (spoke) hospital (drip-and-stay) or transferring patients to a central treating (hub) hospital (drip-and-ship) is not established. We sought to compare outcomes between drip-and-ship and drip-and-stay patients treated with tPA via telemedicine. We hypothesized that there would be no differences in short-term outcomes of in-hospital mortality, length of stay, or discharge disposition or in 90-day outcomes between groups. Methods- We retrospectively identified patients treated with tPA at 17 spoke hospitals between September 2015 and December 2016. Demographic, clinical, and outcome data were obtained from a prospective telemedicine registry. We used negative binomial, multinomial, and logistic regression analyses to evaluate length of stay, discharge disposition, and inpatient mortality, respectively. We compared the proportion of patients with 90-day modified Rankin Scale score <2 by group. Results- Among 430 tPA-treated patients, 232 (53.9%) were transferred to the hub after treatment. The median arrival National Institutes of Health Stroke Scale score was higher for drip-and-ship (10; interquartile range, 5-18) compared with drip-and-stay patients (6; interquartile range, 4-10; P<0.001). Unadjusted length of stay was longer in drip-and-stay patients (incidence rate ratio, 0.82; 95% CI, 0.71-0.95). There were no significant differences in adjusted length of stay, hospital mortality, or discharge disposition. Among the 64% of patients with complete 90-day modified Rankin Scale score, the proportion with good outcomes (modified Rankin Scale score <2) did not differ between groups. Conclusions- We found no differences in measured outcomes between drip-and-ship and drip-and-stay patients treated in our network, although our study may be underpowered to detect small differences.
DOI:10.17219/acem/84936URLPMID:30507073 [本文引用: 2]
BACKGROUND: Post-thrombotic intracerebral hemorrhage (ICH) is experienced by 6-8% of stroke patients and is associated with multiple factors, including acquired coagulopathy induced by the thrombolytic drug. OBJECTIVES: The objective of this study was to assess the outcome of the intravenous (IV) administration of fibrinogen concentrate in a series of acute stroke patients who developed iatrogenic fibrinogen critical depletion after IV thrombolysis. MATERIAL AND METHODS: Of the 39 ischemic stroke patients treated with IV thrombolysis with a severe hypofibrinogenemia requiring infusion with IV fibrinogen concentrate, 30 patients were treated with 2 g of IV recombinant tissue plasminogen activator (rt-PA), followed by further doses until the fibrinogen level reached 200 mg/dL in hemorrhagic patients or 100 mg/dL in non-hemorrhagic patients, and 9 were treated with IV rt-PA followed by endovascular thrombectomy. RESULTS: Preand post-thrombolysis National Institutes of Health Stroke Scale (NIHSS) scores were statistically different for the Cochran-Mantel-Haenszel test overall (p = 0.0002), at 24-hour evaluation (p = 0.0455) and at 7-day assessment (p = 0.0006). Within the first 7 days post-thrombolysis, the brain computed tomography (CT) scans showed that 20/39 (51.28%) patients had ICH. Of the whole sample, 25.6% of the ICH patients had symptomatic intracerebral hemorrhage (SICH), according to National Institute of Neurological Disorders and Stroke (NINDS) classification. After rt-PA treatment, the median pre-thrombolysis fibrinogenemia of 332 mg/dL significantly dropped to 133 mg/dL (p < 0.0001). After the fibrinogen concentrate infusion, the median level of fibrinogenemia rose to 160 mg/dL, which was significantly higher than the median postthrombolysis levels (p < 0.0001). Recanalization was observed in 25/28 patients (89.29%): complete in 18 and partial in 7 patients. After fibrinogen IV infusion, no thrombotic complications were seen in 37 out of 39 patients (94.77%); 2/39 (0.05%) patients experienced a pulmonary embolism, 1 of them a segmental one. CONCLUSIONS: This study showed the clinical safety of administering IV fibrinogen concentrate in order to increase plasma fibrinogen levels in a series of acute stroke patients with iatrogenic fibrinogen depletion after IV thrombolysis.
URL [本文引用: 2]
URL [本文引用: 2]
DOI:10.1016/j.ymthe.2019.08.006URLPMID:31494053 [本文引用: 1]
Our goal is the development of in vivo hematopoietic stem cell (HSC) transduction technology with targeted integration. To achieve this, we modified helper-dependent HDAd5/35++ vectors to express a CRISPR/Cas9 specific to the
URLPMID:27445863 [本文引用: 1]
[本文引用: 1]
[本文引用: 1]
URL [本文引用: 2]
URL [本文引用: 2]
URL [本文引用: 2]
[本文引用: 2]
URLPMID:16013479 [本文引用: 1]
Gene chip technology was employed to study gene expression in Tamarix androssowii under NaHCO3 stress. cDNAs from T. androssowii treated with NaHCO3 solution and that from control group were labeled with fluorescent dye CyS and Cy3 respectively. The two fluorescent cDNA probes were mixed and hybridized to gene chips containing T. androssowii genes, and the chips were scanned using biochip scanning system. Differential expression of genes was analyzed through calculation of the ratio of Cy5 to Cy3 signal intensities. Total of 89 genes differentially expressed were identified, among them, 27 showed down regulated expression and 62 showed up regulated expression. Blastx analysis showed that the function of the differentially expressed genes could be grouped into some categorizations such as photosynthesis, reactive oxygen species eliminated, regulation of osmotic potential, regulation of gene expression and signal transduction, metabolism, development, ribosomal protein, protein breakdown and recycling, transporter, water channel proteins and so on. Based on this research, some function-unknown or novel unreported genes that respond to salt stress were also identified, and these genes may have important functions in salt resistance of T. androssowii. Some important pathways of salt resistance in T. androssowii are revealed, and the gene expression profiling of T. androssowii under salt stress and without stress is obtained in this study.
URLPMID:16013479 [本文引用: 1]
Gene chip technology was employed to study gene expression in Tamarix androssowii under NaHCO3 stress. cDNAs from T. androssowii treated with NaHCO3 solution and that from control group were labeled with fluorescent dye CyS and Cy3 respectively. The two fluorescent cDNA probes were mixed and hybridized to gene chips containing T. androssowii genes, and the chips were scanned using biochip scanning system. Differential expression of genes was analyzed through calculation of the ratio of Cy5 to Cy3 signal intensities. Total of 89 genes differentially expressed were identified, among them, 27 showed down regulated expression and 62 showed up regulated expression. Blastx analysis showed that the function of the differentially expressed genes could be grouped into some categorizations such as photosynthesis, reactive oxygen species eliminated, regulation of osmotic potential, regulation of gene expression and signal transduction, metabolism, development, ribosomal protein, protein breakdown and recycling, transporter, water channel proteins and so on. Based on this research, some function-unknown or novel unreported genes that respond to salt stress were also identified, and these genes may have important functions in salt resistance of T. androssowii. Some important pathways of salt resistance in T. androssowii are revealed, and the gene expression profiling of T. androssowii under salt stress and without stress is obtained in this study.
[本文引用: 1]
[本文引用: 1]
[本文引用: 1]
[本文引用: 1]
DOI:10.1007/s11033-016-4020-0URLPMID:27230577 [本文引用: 5]
The human tissue-type plasminogen activator (tPA) is a key kinase of fibrinolysis that plays an important role in dissolving fibrin clots to promote thrombolysis. The recombinant human plasminogen activator (rhPA) has more thrombolytic advantages than the wild type tPA. To increase the half-life and thrombolytic activity of tPA, a mutant containing only the essential K2 fibrin-binding and P activating plasminogen domains of the wild type tPA was cloned. This fragment was then inserted into goat beta-casein regulatory sequences. Then, a mammary gland-specific expression vector, PCL25/rhPA, was constructed, and the transgenic rabbits were generated. In this study, 18 live transgenic founders (12female symbol, 6male symbol) were generated using pronuclear microinjection. Six transgenic rabbits were obtained, and the expression levels of rhPA in the milk had a range of 15.2-630 microg/ml. A fibrin agarose plate assay of rhPA showed that it had strong thrombolytic bioactivity in vitro, and the highest specific activity was >360 (360 times more than that of alteplase). The results indicated that the rhPA containing only the K2 and P domains is efficiently expressed with higher thrombolytic bioactivity in the milk of transgenic rabbits. Our study also demonstrated a new method for the large-scale production of clinically relevant recombinant pharmaceutical proteins in the mammary glands of transgenic rabbits.
DOI:10.5713/ajas.16.0965URLPMID:28728377 [本文引用: 1]
OBJECTIVE: In the present study, we examined whether the post-prandial reduction in plasma growth hormone (GH) levels is related to the increase in plasma insulin levels in ruminants. METHODS: We performed two experiments: intravenous bolus injection of insulin (0.2 IU/kg body weight) or glucose (1.0 mmol/kg body weight) was administered to increase the plasma insulin levels in male Shiba goats. RESULTS: In the insulin injection experiment, significant (p<0.05) increase in GH concentrations was observed, 15 to 20 min after the injection; it was accompanied with a significant (p<0.01) increase in cortisol concentrations at 45 to 90 min, when compared to the concentrations in the saline-injected controls. The glucose injection significantly (p<0.05) increased the plasma GH concentration at 20 to 45 min; this was not accompanied by significantly higher cortisol concentrations than were observed for the saline-injected control. Hypoglycemia induced by the insulin injection, which causes the excitation of the adrenal cortex, might be involved in the increase in insulin levels. CONCLUSION: Based on these results, we conclude that post-prandial increases in plasma insulin or glucose levels do not induce a decrease in GH concentration after feeding in the ruminants.
DOI:10.33549/physiolres.933715URLPMID:29303609 [本文引用: 2]
Dairy goats are often fed a high-concentrate (HC) diet to meet their lactation demands; however, long-term concentrate feeding is unhealthy and leads to milk yield and lactose content decreases. Therefore, we tested whether a buffering agent is able to increase the output of glucose in the liver and influence lactose synthesis. Eight lactating goats were randomly assigned to two groups: one group received a HC diet (Concentrate : Forage = 6:4, HG) and the other group received the same diet with a buffering agent added (0.2 % NaHCO(3), 0.1 % MgO, BG) over a 19-week experimental period. The total volatile fatty acids and lipopolysaccharide (LPS) declined in the rumen, which led the rumen pH to become stabile in the BG goats. The milk yield and lactose content increased. The alanine aminotransferase, aspartate transaminase, alkaline phosphatase, pro-inflammatory cytokines, LPS and lactate contents in the plasma significantly decreased, whereas the prolactin and growth hormone levels increased. The hepatic vein glucose content increased. In addition, pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6PC) expression in the liver was significantly up-regulated. In the mammary glands, the levels of glucose transporter type 1, 8, 12 as well as of sodium-glucose cotransporter 1 increased. Cumulative buffering agent treatment increased the blood concentrations of glucose via gluconeogenesis and promoted its synthesis in the liver. This treatment may contribute to the increase of the milk yield and lactose synthesis of lactating goats.
[D].
[本文引用: 4]
[D].
[本文引用: 4]
URLPMID:29212747 [本文引用: 2]
DOI:10.1007/s10529-017-2285-6URLPMID:28091772 [本文引用: 1]
OBJECTIVE: To use a transient expression system to express a truncated human tissue plasminogen activator (K2S) gene in cucurbit plants. RESULTS: The recombinant tissue plasminogen activator protein (K2S form) was expressed in active form in cucurbit plants. Its molecular weight was 43 kDa. The plant-derived rt-PA was determined using goat anti-rabbit antibody by western blotting. Among the infected lines, the highest expression of rt-PA was 62 ng/100 mg per leaf tissue as measured by ELISA. The enzymatic activity of the plant-derived rt-PA was 0.8 IU/ml. CONCLUSIONS: The K25 form of rt-PA was expressed for the first time using the viral expression system. Plant-derived rt-PA showed similar potency to commercially-available PA.
[本文引用: 1]
URLPMID:30118482 [本文引用: 1]
[本文引用: 1]
DOI:10.1111/apa.13932URLPMID:28543706 [本文引用: 1]
AIM: Children treated with a growth hormone (GH) for idiopathic growth hormone deficiency (IGHD) may be monitored with the first-year prediction model from the Pfizer International Growth Database (KIGS) using auxology, age, GH dose and the maximum GH concentration from a stimulation test (GHmax stim). We tested the hypothesis that using a 12-hour spontaneous profile (GHmax 12h) would be as accurate. METHODS: We studied 98 prepubertal Swedish children (78 boys) aged 2-12 years enrolled in KIGS. The first-year growth was predicted using the GHmax from the GH profile and a stimulation test, and both of these were compared separately with the observed growth response. RESULTS: The increased height observed in the first year was 0.74 standard deviation scores (SDS), and the studentised residuals for the predicted and observed growth with GHmax stim (-0.16 SDS) and GHmax 12h (-0.22) were similar. Individual predictions calculated with stimulated or spontaneous GHmax showed a significant correlation (r = 0.80). CONCLUSION: We validated the KIGS IGHD prediction model and found that the stimulated GHmax peak can be reliably replaced by the GHmax 12h with similar accuracy. This makes the model more accessible for clinicians, who can then provide realistic expectations for the growth response during the first year of treatment.
DOI:10.3168/jds.2007-0509URLPMID:18096930 [本文引用: 1]
Growth hormone (GH) can increase milk production in cattle, and this effect was thought to be mediated by an indirect mechanism because traditional ligand binding assays failed to detect GH binding sites in the mammary gland. However, recent findings that GH receptor (GHR) mRNA and protein are expressed in the epithelial cells of the bovine mammary gland suggest that GH may directly act on these cells to affect milk production. Therefore, the objective of this study was to determine whether GH could affect milk protein gene expression, nutrient uptake, and cell proliferation in bovine mammary epithelial cells using the bovine mammary epithelial cell-derived MAC-T cells as a model. Native MAC-T cells had low expression of GHR. Thus, we transfected them with expression plasmids for GHR and signal transducer and activator of transcription 5 (STAT5), 2 key components of GHR signaling, to maximize their GH response. Growth hormone increased the expression of alphaS1-casein, alphaS2-casein, beta-casein, and alpha-lactalbumin mRNA 16- to 117-fold in the transfected MAC-T cells, whereas it had no effect on the expression of kappa-casein, beta-lactoglobulin, or insulin-like growth factor I mRNA. Cotransfection analyses showed that GH also strongly induced reporter gene expression from alphaS1-casein, alphaS2-casein, beta-casein, and alpha-lactalbumin gene promoters. Growth hormone had no effect on the uptake of 2-deoxyglucose, an unmetabolizable glucose analog, amino acids, or oleic acid; neither did it affect cell proliferation or death. These observations together with the fact that GH receptor mRNA and protein are expressed in the epithelial cells of the bovine mammary gland raise the possibility that GH might act directly on the mammary epithelial cells in cows to stimulate transcription of major milk protein genes, as part of the mechanism by which GH stimulates milk production.
[本文引用: 1]
[本文引用: 1]
URLPMID:3892305 [本文引用: 1]
URL
URL
[本文引用: 1]
[本文引用: 1]
DOI:10.1126/science.6356363URLPMID:6356363 [本文引用: 1]
The promoter or regulatory region of the mouse gene for metallothionein-I was fused to the structural gene coding for human growth hormone. These fusion genes were introduced into mice by microinjection of fertilized eggs. Twenty-three (70 percent) of the mice that stably incorporated the fusion genes showed high concentrations of human growth hormone in their serum and grew significantly larger than control mice. Synthesis of human growth hormone was induced further by cadmium or zinc, which normally induce metallothionein gene expression. Transgenic mice that expressed human growth hormone also showed increased concentrations of insulin-like growth factor I in their serum. Histology of their pituitaries suggests dysfunction of the cells that normally synthesize growth hormone. The fusion genes were expressed in all tissues examined, but the ratio of human growth hormone messenger RNA to endogenous metallothionein-I messenger RNA varied among different tissues and different animals, suggesting that expression of the foreign genes is influenced by site of integration and tissue environment.
DOI:10.1016/j.anireprosci.2014.11.005URLPMID:25482591 [本文引用: 1]
Body weight in different mammalian species influences reproductive potential. The aim of the present study was to determine the relationship of body weight at the time of semen collection with libido, seminal characteristics and number of semen doses for artificial insemination (AI) in New Zealand White mature fertile male rabbits. Data came from 728 semen collections of 14 rabbits, 15-months of age that were sexually experienced with proven semen quality and fertility. Semen collection was performed twice a week with two ejaculates at each collection time and lasted 14 weeks. A second ejaculation was collected at 1-2h after the first. Data from each male from first and second ejaculates from 1 day of semen collection throughout the trial were averaged (n=324) and partial correlation coefficients and regression equations were estimated to describe the relationship of male body weight to ejaculation reaction time and 12 semen and sperm characteristics. As body weight increased there was a linear (P<0.05) increase in reaction time, abnormal sperm with an intact membrane and abnormal sperm with a damaged membrane and a linear (P<0.05) decrease in semen volume, sperm concentration per ejaculate, normal sperm with an intact membrane, number of normal motile sperm with an intact membrane and suitable semen doses for AI. Body weight of the mature male rabbit at semen collection had some influence on libido, semen and sperm characteristics, with a general trend toward a lesser reproduction potential as body weight increases.
[本文引用: 1]
