王嘉琪^,1, 董育红¹, 姜菊玲¹, 钱建宁¹, 魏文涛¹, 宋国亮², 焦金波², 关新新², 姬郭彪², 张业炘¹¹甘肃健顺生物科技有限公司,兰州730070
²洛阳惠中生物技术有限公司,河南洛阳471000

Based on PK15 Cell Line for PCV2 Fully Suspension Culture Process

WANG JiaQi^,1, DONG YuHong¹, JIANG JuLing¹, QIAN JianNing¹, WEI WenTao¹, SONG GuoLiang², JIAO JinBo², GUAN XinXin², JI GuoBiao², ZHANG YeXin¹¹Gansu Jianshun Biotechnology Co., Ltd, Lanzhou 730070
²Luoyang Huizhong Biotech Co.,Ltd., Luoyang 471000, Henan

责任编辑: 林鉴非
收稿日期:2020-03-20接受日期:2020-06-30网络出版日期:2021-03-16

基金资助:

兰州市2019年度重点人才项目

Received:2020-03-20Accepted:2020-06-30Online:2021-03-16
作者简介 About authors
王嘉琪,E-mail：wangjiaqi@jianshunbio.com












摘要
【目的】筛选1株适合PCV2病毒生产的悬浮细胞株,摸索PCV2悬浮生产工艺（病毒种毒来源、MOI及收获时间）,为大规模悬浮培养技术制备疫苗提供试验依据。【方法】使用有限稀释法对PK15原细胞稀释后接种于96孔板,每2 d观察细胞生长和形态,待细胞90%长满后,将细胞从96孔板陆续扩至24孔板、12孔板、6孔板,最后到方瓶,筛选3株可贴壁生长的、形态较好的PK15克隆。3株克隆（PK15-1C8、2F11、1E5）按照1×10⁶细胞/mL的密度,直接接种在PK15的无血清培养基中,并置于37℃,5%二氧化碳,120 r/min的摇床培养箱中继续培养,每天监测细胞密度和活率,每3 d传代,使细胞逐渐适应悬浮环境,驯化为可全悬浮、无血清培养的悬浮细胞株;细胞传代稳定并建库后,接种PCV2病毒,通过对比3株悬浮克隆细胞培养PCV2病毒含量的差异,筛选1株克隆细胞,用于生产PCV2;针对不同种毒（来源于贴壁细胞或悬浮细胞）,摸索感染MOI（0.1、0.2、0.5）及收获时间（48、72、96、120h）,确定PCV2最佳生产工艺。【结果】（1）贴壁细胞置于无血清培养基中,适应至第2代时细胞即可呈悬浮生长,连续传至11代,细胞生长稳定,按照1×10⁶/mL接种细胞,细胞生长72h时细胞密度可达到10×10⁶/mL,活率在95%以上,倍增时间为20h左右;（2）3株悬浮细胞使用相同条件,分别接种PCV2病毒后,PK15-1C8克隆细胞的病毒含量可达到10^6.4TCID₅₀/mL,克隆PK15-2F11（10^5.5TCID₅₀/mL）、PK15-1E5（10^5.6TCID₅₀/mL）,3株克隆细胞病毒含量均高于原克隆（10^4.7TCID₅₀/mL）,但PK15-1C8克隆细胞的病毒含量更高且更稳定,确定为后续研究用细胞;（3）使用贴壁细胞来源的种毒（10^6.4TCID₅₀/mL）感染PK15-1C8克隆细胞后,最优工艺为接毒时细胞密度1×10⁶/mL,以0.1MOI接毒,接毒后72h收获,最高病毒含量为10^6.5TCID₅₀/mL。以来源于悬浮细胞的种毒（10^6.3TCID₅₀/mL）感染细胞后,病毒含量较贴壁细胞来源种毒更高,最高可达10^7.3TCID₅₀/mL,其最优工艺为接毒时细胞密度1×10⁶/mL,以0.2MOI接毒,接毒后72h收获。【结论】通过对PK15细胞进行单克隆筛选,驯化悬浮、3株悬浮细胞接种PCV2后病毒含量的对比,确定一株病毒含量最高的悬浮细胞,并以此悬浮细胞为基础进行PCV2生产工艺摸索,建立了全悬浮无血清培养的PK15-1C8细胞增殖PCV2工艺,该工艺首次使用悬浮细胞扩增PCV2病毒为种毒进行接毒,最高病毒含量可达10^7.3TCID₅₀/mL,可用于工厂化疫苗生产。
关键词： PK15细胞;猪圆环病毒2型;克隆筛选;悬浮培养

Abstract
【Objective】Selected one suspension PK15 cell line which is suitable for PCV2 virus , and then developed the production process for PCV2 vaccines(source of virus, MOI and harvest time ), to provide basic theory and guarantee for large-scale production use suspension culture instead of adherent culture. 【Method】The PK15 primary cells were diluted using the limiting dilution method and seeded in 96-well plates. The cell growth and morphology were observed every 2 days. After 90% of the cells were overgrown, the cells were gradually expanded from 96-well plates to 24-well plates,to 12-well plates, to 6-well plate, and finally to the square flask, three PK15 clones with good morphology that can grow adherently were selected. Three PK15 clones with good morphology that can grow adherently were selected. Three clones (PK15-1C8, 2F11, 1E5) were directly seeding in PK15 serum-free medium at a density of 1×10⁶cells/mL and placed in a shaker incubator at 37 ℃, 5% carbon dioxide, and 120 r/min to continue culture. Monitor the cell density and viability every day, passage every 3 days, make the cells gradually adapt to the suspension environment, the PK15 clone can culture as a fully suspension cell line and can growth well in serum free media. After suspension cells stability passage and cell bank, compared the PCV2 virus content with three suspension clone cells, one clone cell was selected for PCV2 production. For different kinds of viruses (source from adherent cells or suspension cells), explore the infection MOI (0.1, 0.2, 0.5) and harvest time (48, 72, 96, 120h), determine the best production process of PCV2. 【Results】(1)The results showed that when the adherent cells passage to second generation in serum media CD PK15 259, the cells can suspended growth, continuous passage for eleven generation, suspension cells can growth stable, when seeding with 1×10⁶ /mL cells, cells can reach 10×10⁶ /mL cells when growth at 72h, viability rate is above 95%, and the doubling time is about 20h; (2) The three suspension cells use the same condition to infection PCV2 virus, the virus content of PK15-1C8 cloned cells can reach 10^6.4TCID₅₀/mL, clone PK15-2F11 (10^5.5TCID₅₀/mL), PK15-1E5 (10^5.6TCID₅₀/mL), the virus content of the three cloned cells was higher than that of the original clone (10^4.7TCID₅₀/mL), however, the virus content of PK15-1C8 cloned cells is higher and more stable, so it is determined as a cell for follow-up research; (3) After infecting PK15-1C8 cloned cells with seed virus (10^6.4TCID₅₀ /mL) from adherent cells, the optimal process is 1×10⁶/mL cell density with 0.1MOI and harvest at 72h the virus content can reach 10^6.5TCID₅₀ /mL. After infecting PK15-1C8 cloned cells with seed virus (10^6.3TCID₅₀ /mL) from suspension cells, the optimal process is 1×10⁶/mL cell density with 0.2MOI and harvest at 72h the virus content can reach 10^7.3TCID₅₀ /mL. 【Conclusion】Through the monoclonal screening of PK15 cells, adapted to suspension cells compared the PCV2 virus content of 3 suspension cells, a suspension cell with the highest virus content was determined, and based on this suspension cell, explored the process of PCV2 virus, and then established the PCV2 fully suspension, serum-free culture process. The SV15-1C8 cell proliferation PCV2 process of full suspension serum-free culture was established. This process used suspension cells to amplify PCV2 virus for seed poisoning for the first time. The highest virus content can reach 107.3TCID50/mL, which can be used for factory vaccine production. According selection sensitive clones and optimized the process for PCV2, can increase the virus titer, and realizes full suspension culture without serum, improved the production process of PCV2, improve the production efficiency, reduce the cost and improve the quality of the production. This process first use suspension cells to amplify the PCV2 virus. The highest virus content can reach 10^7.3TCID₅₀/mL, which can be used for large-scale PCV2 virus production.
Keywords：PK15 cells;PCV2;clone selection;suspension culture


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王嘉琪, 董育红, 姜菊玲, 钱建宁, 魏文涛, 宋国亮, 焦金波, 关新新, 姬郭彪, 张业炘. 基于PK15细胞的猪圆环病毒2型全悬浮培养工艺[J]. 中国农业科学, 2021, 54(6): 1280-1287 doi:10.3864/j.issn.0578-1752.2021.06.017
WANG JiaQi, DONG YuHong, JIANG JuLing, QIAN JianNing, WEI WenTao, SONG GuoLiang, JIAO JinBo, GUAN XinXin, JI GuoBiao, ZHANG YeXin. Based on PK15 Cell Line for PCV2 Fully Suspension Culture Process[J]. Scientia Acricultura Sinica, 2021, 54(6): 1280-1287 doi:10.3864/j.issn.0578-1752.2021.06.017


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0 引言

【研究意义】猪圆环病毒病（porcine circovirus diseases, PCVDs）是影响养猪业的重要传染病,其病原为猪圆环2型（porcine circovirus type 2, PCV2）。PCV2可引起断奶仔猪多系统衰竭综合症,还与皮炎肾病综合症、增生性坏死性肺炎、猪繁殖与呼吸综合征、仔猪传染性先天性震颤等多种疾病相关,但临床上以多系统衰竭综合症最为常见^{[1,2,3,4,5,6]},是严重危害养猪业健康发展的疫情之一^[7,8]。猪肾细胞（PK15）是目前用于PCV2病毒培养的主要细胞系,用于增殖PCV2和制备猪疫苗。但由于PCV2病毒的编码区非常有限,对宿主细胞具有高度的依赖性^[9],且在PK15细胞系不同的细胞株上增殖的病毒含量不同, 难以达到制备疫苗的要求^[10-12] ,是制约PCV2全病毒灭活苗的技术瓶颈之一。同时PK15细胞贴壁培养时存在需要血清、放大困难和成本高等问题,需筛选出一株对PCV2敏感,且易于放大和产业化生产的病毒培养工艺。【前人研究进展】PK15细胞现已广泛应用于猪圆环病毒,但由于PCV2毒力弱,且不产生细胞病变,获得高病毒含量PCV2难度较大,加之PK15细胞贴壁培养时存在血清,而且放大困难,成本高等问题,因此PCV2的培养病毒含量高低已成为制约现有疫苗质量的关键瓶颈之一^[13,14]。刘俊斌等^{[15,16,17,18]}报道了使用片状载体和微载体在生物反应器中培养PK15贴壁细胞并感染PCV2的工艺研究,通过优化微载体使用浓度、细胞接种密度、细胞初始搅拌方式、生长阶段搅拌速度、培养基补给方式、接毒剂量和收获时间等关键技术参数,提高PCV2产量,确定了反应器生产PCV2的最佳工艺;彭伍平等^[19,20,21]报道了通过筛选贴壁单克隆,找到一株对PCV2敏感的克隆株,从而提高了PCV2病毒含量;刘天伦等 ^[22,23]报道了经贴壁降血清、低血清悬浮优化传代、无血清悬浮传代培养,对一株贴壁PK15细胞进行了无血清全悬浮驯化,并可稳定传代,以期为传代细胞系低血清驯化培养及圆环病毒在低血清培养细胞上的增殖研究奠定基础;李雨慈^[24]报道使用昆虫细胞基因表达技术,首次使用悬浮培养生产的猪圆环病毒病疫苗;郭玲花等^{[25,26,27,28]}报道了猪圆环病毒2型悬浮培养工艺的研究进展,对目前国内外市场上PCV2疫苗的制备工艺及其优缺点进行对比分析,可利用全悬浮培养工艺培养PK-15细胞来增殖PCV2;此外,未见全悬浮培养工艺中以悬浮细胞制备种毒的研究报道。【本研究切入点】筛选出1株PCV2高产克隆细胞株,并建立起无血清全悬浮病毒培养工艺。【拟解决的关键问题】通过全悬浮细胞培养技术,制备出病毒含量高和适合猪圆环病毒病灭活疫苗规模化生产工艺。

1 材料与方法

1.1 材料

1.1.1 试验时间和地点 试验于2018年12月至2019年12月在甘肃健顺生物科技有限公司实验室进行。

1.1.2 细胞株 试验所用细胞株为PK15（猪圆环病毒1型及2型阴性）[PK15, PK-15]ATCC? CCL-33?,甘肃健顺生物科技有限公司保存。

1.1.3 病毒株 PCV2（猪圆环2型病毒分离株,PK15贴壁获得P14病毒,病毒含量10^6.4TCID₅₀/mL,支原体检验、BVDV、CSFV、致细胞病变检查及红细胞吸附性外源病毒检验均为阴性）由洛阳惠中生物技术有限公司提供。

1.1.4 主要试剂和耗材 DMEM高糖、CD PK15 259（PK15悬浮细胞生长培养基）、PK15接毒培养基、0.25%胰蛋白酶-EDTA、接毒培养基A、接毒培养基B（以上试剂均由甘肃健顺生物科技有限公司提供）,FBS（兰州荣晔生物科技有限公司）,PCV2一抗、羊抗鼠二抗（南京华恩生物科技有限公司）,荧光显微镜（OLYMPUS）,Vi-Cell细胞计数仪。

1.2 方法

1.2.1 贴壁PK15细胞克隆 采用有限稀释法,将细胞铺于96孔培养板,于37℃、5%CO₂培养箱中培养,第4天于显微镜下初步挑选单克隆,每两天显微镜下观察,保证孔中细胞为单克隆团,将克隆细胞逐步放大培养。

1.2.2 克隆细胞株悬浮驯化 待细胞扩大至T225瓶时,消化,计数,离心,采用PK15悬浮生长培养基CD PK15 259按1.0×10⁶个细胞/mL密度重悬细胞,并于37℃、5%CO₂摇床培养箱中培养,至细胞适应悬浮培养基可正常生长。

1.2.3 PCV2高产细胞筛选 待驯化悬浮的PK15-2F11、1E5、1C8细胞生长至第3天时,分别使用PK15接毒培养基A和PK15接毒培养基B将细胞稀释至1.0×10⁶个细胞/mL,工作体积30mL分别接于125mL摇瓶中,按体积比5%加入PCV2病毒,37℃、5%CO₂于摇床培养箱中培养,每天取样计数,72h后收毒,采用测定病毒含量。并将收获病毒液,对应细胞第二次感染各克隆细胞。

1.2.4 接毒工艺优化 接毒培养基A将培养至第3天的PK15-1C8细胞稀释至1.0×10⁶个细胞/mL,按0.1、0.2、0.5MOI（PCV2来源于贴壁细胞毒）接毒,试验重复3次,或按0.1、0.2、0.5MOI（PCV2来源于悬浮细胞毒）接毒,试验重复5次,不同时间段收获病毒液,测定病毒含量（表1）。

Table 1
表1
表1接毒工艺优化试验参数
Table 1Experiment parameters for optimization of infection process

试验 Experiment		感染培养基 Infection medium	感染时细胞密度 Infection cell density（cells/mL）	感染量 MOI	收获时间 Harvest time （h）	重复次数 Repeat times
贴壁种毒 Virus from adherent cell	感染MOI摸索 Infection MOI explore	接毒培养基A Infection medium A	1.0×106	0.1、0.2、0.5	72	3
	收获时间摸索 Harvest time explore		1.0×106	0.1	48、72、96、120	3
悬浮种毒 Virus from suspension cell	感染MOI摸索 Infection MOI explore	接毒培养基A Infection medium A	1.0×106	0.1、0.2、0.5	72	5
	收获时间摸索 Harvest time explore		1.0×106	0.2	48、72、96	5

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1.2.5 病毒含量测定 用含2%血清的DMEM培养基将所取病毒液作10倍系列稀释,取10^-4、10^-5、10^-6、10^-7 4个稀释度,分别接种在单层PK15细胞的96孔细胞培养板中,每个稀释度接种6孔,100μL /孔,同时设正常细胞对照组,采用Reed-Müench法计算TCID₅₀。

2 结果

2.1 PK15单克隆细胞驯化悬浮

将挑出来的4个单克隆贴壁细胞及PK15原细胞株驯化悬浮,成功驯化3株克隆和PK15细胞,细胞生长曲线如图1所示,PK15、PK15-2F11和1C8 3株细胞均1代便从贴壁细胞驯化为悬浮细胞,且细胞生长稳定,PK15-1E5前5代生长较慢,6代后细胞生长变快渐渐生长稳定。4株细胞生长稳定之后,3d生长情况相似,密度几乎都为10×10⁶个细胞/mL,PK15、PK15-2F11、1E5、1C8细胞倍增时间为分别为21、21、20和21 h。

图1

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图14株单克隆细胞驯化悬浮生长曲线

A.PK15细胞驯化生长曲线。B.PK15-2F11细胞驯化生长曲线。C.PK15-1E5细胞驯化生长曲线。D. PK15-1C8细胞驯化生长曲线。其中,实线为细胞生长密度曲线,虚线为细胞活率曲线。浅色线为3种单克隆和PK15贴壁细胞生长曲线,深色线条为3种单克隆和PK15细胞驯化悬浮细胞生长曲线。
Fig. 1Adaption suspension growth curves of four monoclonal cells

A. PK15 cells adapted growth curve. B. Acclimation growth curve of PK15-2F11 cells. C. Acclimation growth curve of PK15-1E5 cells. D. Acclimation growth curve of PK15-1C8 cells. Among them, the solid line is the cell growth density curve, and the dashed line is the cell viability curve. The light-colored line is the growth curve of 3 kinds of monoclonal and PK15 adherent cells, and the dark line is the growth curve of the acclimated suspension cells of 3 kinds of monoclonal and PK15 cells.

2.2 PCV2敏感细胞株筛选

将PK15细胞及PK15-2F11、PK15-1E5、PK15-1C8 4株细胞感染PCV2（种毒由悬浮细胞获得）,TCID₅₀结果如图2-A所示,3株克隆细胞毒价均高于原始细胞株,PK15-1C8试验组较其他2株克隆细胞毒价更高,病毒含量为10^6.0TCID₅₀/mL,使用接毒培养基A接毒组（PK15-1C8-C1）与使用接毒培养基B接毒组（PK15-1C8-C2）病毒含量无差异。为了检测第一次试验结果的可靠性,将第一次所收获病毒液,对应细胞株进行第二次接毒（图2-B）,3株克隆细胞感染PCV2后仍然是PK15-1C8更高,是2F11、1E5的10倍,病毒含量维持在10^6.0TCID₅₀/mL。

图2

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图2PK15细胞感染PCV2后TCID₅₀

A.PK15、PK15-2F11、PK15-1E5、PK15-1C8四株细胞分别感染PCV2后TCID₅₀。B.PK15-2F11、PK15-1E5、PK15-1C8三株细胞分别对应感染图A收获PCV2病毒。其中,C1为使用接毒培养基A接毒实验组,C2为使用接毒培养基B接毒实验组。
Fig. 2TCID₅₀ after PCV2 infection in PK15 cells

A. TCID₅₀ of PK15, PK15-2F11, PK15-1E5, PK15-1C8 cells infected with PCV2. B. PK15-2F11, PK15-1E5, and PK15-1C8 were infected with PCV2 virus harvest from Figure A. Among them, C1 is the experimental group infected with inoculation medium A, and C2 is the experimental group infected with inoculation medium B.

2.3 接毒工艺优化

2.3.1 贴壁细胞收获病毒为感染种毒工艺优化 如表1所示进行试验,不同MOI感染细胞重复3次试验后,3种不同MOI感染的细胞毒价差异不大均在10^6.5TCID₅₀/mL左右（表2）,因此选取病毒添加较少的0.1MOI接毒。

Table 2
表2
表2不同MOI、不同时间的PCV2感染PK15-1C8病毒含量
Table 2The content of PK15-1C8 virus infected with PCV2 at different MOI and different time

参数 Parameter	条件 Condition	实验中病毒含量（lgTCID50/mL） Virus content（lgTCID50/mL）
		1	2	3
感染病毒量 MOI	0.1	6.5	6.6	6.0
	0.2	6.7	6.4	6.2
	0.5	6.3	6.5	6.2
取样时间/h Sample Time/h	48	6.4	5.8	6.4
	72	6.5	6.3	6.3
	96	6.7	6.4	5.6
	120	6.7	6.7	5.6

新窗口打开|下载CSV

选取接毒计量(MOI)为0.1的感染条件,于培养48、72、96、120 h取样,检测病毒含量,综合3次试验结果72 h收获病毒含量lgTCID₅₀/mL较高且更稳定。

2.3.2 悬浮细胞收获病毒为感染种毒来源相关工艺确认 接毒计量及收毒时间摸索, 如表3所示,分别按0.1、0.2、0.5MOI将由悬浮PK15细胞所获PCV2病毒感染PK15-1C8细胞,0.2MOI感染后,病毒含量更稳定。因此当种毒来源于悬浮细胞时,0.2MOI接毒更佳。

Table 3
表3
表3不同MOI、不同时间的PCV2感染PK15-1C8病毒含量
Table 3The content of PK15-1C8 virus infected with PCV2 at different MOI and different time

参数 Parameter	条件 Condition	实验中病毒含量（lgTCID50/mL） Virus content（lgTCID50/mL）
		1	2	3	4	5
感染病毒量 MOI	0.1	6.5	6.1	6.2	6.0	6.0
	0.2	7.0	6.7	6.5	6.3	6.3
	0.5	6.8	6.7	6.3	5.5	5.7
取样时间 Sample time (h)	48	6.5	6.2	6.4	6.3	6.0
	72	7.2	7.3	6.9	6.5	6.3
	96	6.7	7.2	6.4	6.5	6.3

新窗口打开|下载CSV

以0.2MOI的接毒量,相同试验方法接毒,检测 48、72、96 h病毒含量。48 h病毒含量低于72与96 h,但72与96 h病毒含量差异不大,从病毒含量和生产的时间效率考虑,以悬浮细胞制备的病毒为种毒,接毒量为0.2MOI 72h收获更佳。

3 讨论

PK15细胞作为PCV2主要感染细胞,广泛应用于PCV2疫苗的生产,但由于PCV2 DNA的合成依赖于细胞周期S期表达的酶,随宿主细胞的复制而复制,因此病毒的复制周期更长,使得PCV2体外增值能力较差^[29,30,31]。彭伍平等^[19,20,21]通过对PK15贴壁细胞进行有限稀释后,挑选出的单克隆细胞表现出了对PCV2的高敏感性,提示我们可以通过该方法提高病毒产量,但贴壁细胞生产放大采用的细胞工厂或转瓶工艺,劳动强度大,可使用的细胞密度低^[18],而微载体技术虽然可以有效提高细胞密度,但仍存在生产成本高等弊端,且血清的使用对产品的可控性、安全性、批间差产生一定影响^[22,23]。而悬浮培养细胞,可以在较小的空间内达到较高的细胞密度,同时无血清培养基的使用,使得细胞培养成本低、产品可控、重复性高。因此本文采用有限稀释法挑选出单克隆细胞,并将其驯化悬浮,筛选出对PCV2高敏感的PK15悬浮细胞,以期解决PCV2在生产中病毒含量低的问题。同时通过查阅文献发现,病毒感染时补料的添加、不同感染MOI及收获时间也是影响病毒产量的原因^[16,32],因此在挑选出单克隆悬浮细胞的基础上,进一步进行了工艺优化,确定了最佳的病毒感染MOI及收获时间。

刘天伦等^[22]的研究,通过贴壁细胞逐渐降血清,到悬浮细胞低血清,再到悬浮细胞无血清的过程,经过了23代传代,将PK15贴壁细胞驯化为悬浮细胞,悬浮细胞72h最高生长密度为4.0×10⁶/mL。而本文通过将贴壁PK15使用有限稀释法稀释获得单克隆细胞,并从贴壁细胞含10%血清直接驯化为悬浮无血清,驯化后第二代即可高密度生长,细胞生长稳定,以3d按1﹕10的比例进行扩大,生长密度可达到10× 10⁶/mL,活率均在95%以上。相较刘天伦^[22]等的研究,本文细胞驯化时间更短,且驯化悬浮后的细胞生长稳定倍增时间短,可大大缩短生产周期,为工业的放大生产提供有力支持;同时本文对悬浮细胞进行了单克隆筛选,筛选出的克隆细胞PCV2病毒含量较原克隆从10^5.0TCID₅₀/mL提高至10^6.5TCID₅₀/mL,可大大提高PCV2病毒疫苗的生产效率,但该悬浮克隆细胞PCV2病毒产量仍未达到理想结果,因此我们进一步进行了工艺优化。

福州大北农与成都天邦开发的DBN-SX07 株灭活疫苗,灭活前半成品病毒含量10^5.5TCID₅₀/mL以上;武汉中博,科前生物等开发的WH株灭活疫苗,灭活前半成品病毒含量在10^7.0TCID₅₀/mL以上;江苏南农高科等开发的SH株灭活疫苗,灭活前半成品病毒含量在10^6.0TCID₅₀/mL以上; 哈维科等开发的LG株灭活疫苗,灭活前半成品病毒含量10^5.5TCID₅₀/mL以上^[33]。为了进一步提高该PCV2敏感克隆悬浮细胞的病毒产量,针对该细胞进行了接毒工艺的摸索,确定了不同种毒（来源于贴壁细胞或者悬浮细胞）来源接毒MOI及收获时间,结果发现,使用悬浮细胞种毒、按照0.2MOI接毒72h收获病毒含量从10^6.5TCID₅₀/mL提高至10^7.3TCID₅₀/mL;较传统贴壁工艺和现有的无血清培养基工艺^[15-17,23]相比,收获时间缩短,接毒剂量低且首次接种悬浮细胞制备的种毒,病毒含量提高了10倍,可提高生产效率,缩短生产周期,同时整个生产工艺采用无血清培养,降低生产成本和纯化难度,提高产品质量和批间一致性。此工艺经过多次试验验证,确定了工艺的稳定性,为后期的PCV2病毒疫苗的研究和生产奠定了基础。

4 结论

本研究通过细胞克隆、悬浮驯化、高产细胞株筛选及PCV2生产工艺摸索,建立了全悬浮无血清培养的PK15-1C8细胞增殖PCV2工艺,为疫苗企业采用无血清悬浮培养工艺提供了试验依据。

参考文献 View Option 原文顺序
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[27] 邢海云, 赵建增, 赵亚荣. 猪2型圆环病毒疫苗的研究进展
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[28] FORT M, SIBILA M, ALLEQUZ A. Porcine circovirus type 2 (PCV2) vaccination of conventional pigs prevents viremia against PCV2 isolates of different genotypes and geographic origins
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The efficacy of recently developed porcine circovirus type 2 (PCV2) vaccines has not been tested yet against PCV2 isolates of the two proposed genotypes. In the present work, the efficacy of a subunit vaccine containing PCV2 capsid protein was evaluated by using a challenge model with four different PCV2 isolates of different genotype and geographic origin. The vaccine prevented the development of viremia in all cases as well as significantly decreased nasal and faecal shedding of the virus. Also, the vaccine elicited PCV2-specific neutralizing antibodies to PCV2 even in the presence of maternally derived immunity.

[29] TISCHER I, PETERS D, RASCH R, POCIULI S. Replication of porcine circovirus: induction by glucosamine and cell cycle dependence
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Multiplication of porcine circovirus (PCV) was found to be inducible by treatment of infected cell cultures with 300 mM glucosamine. One day after glucosamine treatment and after growth in fresh medium, an increase in the number of cells containing virus antigen of up to 50 times as compared to mock-treated cultures was observed. Analysis of this phenomenon revealed that replication of PCV DNA was induced. Only aminohexoses but not hexoses and acetylated aminohexoses were efficacious. The course of PCV replication in synchronized cell cultures infected at different periods of the cell cycle showed that PCV DNA synthesis depends on cellular enzymes expressed during S phase growth of cells. However, whereas in cell cultures treated with glucosamine after infection in G0 or during G1, the start of PCV replication was observed during the first S phase after growth stimulation, the latent period in mock-treated cultures lasted until the second S phase. Also in cell cultures transfected with PCV DNA in G0 or during G1 using DEAE-dextran as mediator, PCV replication started during the first S phase after growth release of the cells. From these findings the conclusion is drawn that glucosamine and DEAE-dextran initiate PCV replication by enabling the PCV genome to get entry to the cell nucleus that normally can be achieved only by inclusion in the daughter nuclei at the end of mitosis.

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[32] 张海洋, 吕超超, 肖燕, 孙进忠. 猪圆环病毒2型高滴度培养方法的研究进展
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[33] 李少丽, 李艳丽, 李新华. 猪圆环病毒病疫苗的应用
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