水痘—带状疱疹病毒疫苗株在Vero细胞中传代适应及ELISA检测血清IgG研究

发布时间：2018-08-06 17:21

【摘要】： 水痘是由水痘-带状疱疹病毒(Varicella-Zoster virus ,VZV)引发的世界性传染疾病。为了研制疫苗和有效诊断水痘,本文研究了VZV疫苗株在Vero细胞中传代适应、制备VZV抗原工艺和ELISA方法。将VZV疫苗株在Vero细胞中连续传代培养,传至第9代时,CPE明显增多,病毒滴度从2～3 log PFU/ml提高到"g4.2 log PFU/ml,能在超低温条件下稳定保存,表明毒株获得了适应性,可用于制备VZV抗原。培养条件试验表明,15L瓶旋转培养的适宜MOI(接种比例,logPFU/cell)为0.01,收获时间在60～96h。收获病变细胞,经超声破碎、离心,制备了VZV全病毒ELISA(VAR ELISA)抗原。用Triton X-100裂解,通过Lentil Lectin Sephrose 4B亲和层析纯化,制备了VZV糖蛋白ELISA( gpELISA)抗原,具有VZV特异性糖蛋白。两种抗原的稀释度达1:3000～1:4000。研究了VZV VAR ELISA和gpELISA的反应条件,建立了相应的方法学。两种抗原最适使用稀释度为1:4000,pH7.4的PBS缓冲液为包被液,10%新生牛血清为封闭液,抗原抗体反应时间为120min,酶标抗体工作浓度为1:120,辣根过氧化物酶作用底物为邻苯二胺,酶标抗体反应时间为120min。Vero gpELISA和VAR ELISA抗原加保护剂具有保存稳定性和可重复性。Vero gpELISA检出WHO VZV国际标准血清的抗体滴度较VAR ELISA提高了4倍,检出下限分别为195mIU/ml和781mIU/ml,所得结果与国外成品试剂盒相符。应用抗原,通过筛查,获得了VZV阳性血清,测出的抗VZV IgG几何平均滴度优于人胚肺二倍体2BS细胞制备的VAR ELISA。 VZV疫苗株在Vero细胞中能传代适应、制备毒种,用15L瓶旋转培养可大量制备VZV抗原。建立了Vero细胞制备的VZV IgG VAR ELISA和gpELISA,方法的灵敏度、特异性和可重复性良好。因此,Vero细胞用于VZV诊断制品的研究和生产是可行的,本研究为今后两种试剂盒的标准化和商品化奠定了基础。
[Abstract]:Varicella is a worldwide infectious disease caused by varicella zoster virus (Varicella-Zoster virus). In order to develop vaccine and diagnose varicella effectively, we studied the method of preparing VZV antigen and ELISA method by subculture and adaptation of VZV vaccine strain in Vero cells. The VZV vaccine strain was cultured in Vero cells continuously, and the virus titer was increased from 2 ~ 3 log PFU/ml to "g 4.2 log PFU / ml" at the 9th passage, which indicated that the strain was adaptable. It can be used to prepare VZV antigen. The culture conditions showed that the suitable MOI (inoculation ratio) was 0.01 and the harvest time was 60 ~ 96 h. VZV virus ELISA (VAR ELISA) antigen was prepared by ultrasonic fragmentation and centrifugation. VZV glycoprotein ELISA (gpELISA) antigen was prepared by cleavage of VZV X-100 and purified by Lentil Lectin Sephrose 4B affinity chromatography. The dilution of the two antigens was 1: 3 000 and 1: 4 000. The reaction conditions of VZV VAR ELISA and gpELISA were studied and the corresponding methodology was established. The best PBS buffer with dilution of 1: 4000g pH 7.4 was used as encapsulation solution, 10% newborn bovine serum as blocking solution, the reaction time of antigen and antibody was 120 min, the working concentration of enzyme labeled antibody was 1: 120, and the substrate of horseradish peroxidase was o-phenylenediamine, and the concentration of enzyme labeled antibody was 1: 120, and the substrate of horseradish peroxidase was o-phenylenediamine. The reaction time of 120min.Vero gpELISA and VAR ELISA antigens plus protection agent was stable and reproducible. The antibody titer of WHO VZV international standard serum detected by Vero gpELISA was 4 times higher than that of VAR ELISA. The detection limits were 195mIU/ml and 781 MU / ml, respectively, and the results were in agreement with the foreign product kits. Using antigens, VZV positive serum was obtained by screening. The geometric mean titer of anti VZV IgG was better than that of VAR ELISA. VZV vaccine strain prepared by diploid 2BS cells of human embryo lung. VAR ELISA. VZV vaccine strain could be subcultured and adapted to Vero cells. VZV antigen can be prepared by rotating culture in 15 L bottle. VZV IgG VAR ELISA and gpELISAwere prepared by Vero cells. The sensitivity, specificity and reproducibility of the method were good. Therefore, it is feasible to use Vero cells in the research and production of VZV diagnostic products. This study lays a foundation for the standardization and commercialization of the two kinds of kits in the future.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392;R446.6

【参考文献】