水痘—帶狀皰疹病毒疫苗株在Vero細(xì)胞中傳代適應(yīng)及ELISA檢測血清IgG研究
發(fā)布時間：2018-08-06 17:21
【摘要】： 水痘是由水痘-帶狀皰疹病毒(Varicella-Zoster virus ,VZV)引發(fā)的世界性傳染疾病。為了研制疫苗和有效診斷水痘,本文研究了VZV疫苗株在Vero細(xì)胞中傳代適應(yīng)、制備VZV抗原工藝和ELISA方法。 將VZV疫苗株在Vero細(xì)胞中連續(xù)傳代培養(yǎng),傳至第9代時,CPE明顯增多,病毒滴度從2～3 log PFU/ml提高到"g4.2 log PFU/ml,能在超低溫條件下穩(wěn)定保存,表明毒株獲得了適應(yīng)性,可用于制備VZV抗原。培養(yǎng)條件試驗表明,15L瓶旋轉(zhuǎn)培養(yǎng)的適宜MOI(接種比例,logPFU/cell)為0.01,收獲時間在60～96h。收獲病變細(xì)胞,經(jīng)超聲破碎、離心,制備了VZV全病毒ELISA(VAR ELISA)抗原。用Triton X-100裂解,通過Lentil Lectin Sephrose 4B親和層析純化,制備了VZV糖蛋白ELISA( gpELISA)抗原,具有VZV特異性糖蛋白。兩種抗原的稀釋度達(dá)1:3000～1:4000。 研究了VZV VAR ELISA和gpELISA的反應(yīng)條件,建立了相應(yīng)的方法學(xué)。兩種抗原最適使用稀釋度為1:4000,pH7.4的PBS緩沖液為包被液,10%新生牛血清為封閉液,抗原抗體反應(yīng)時間為120min,酶標(biāo)抗體工作濃度為1:120,辣根過氧化物酶作用底物為鄰苯二胺,酶標(biāo)抗體反應(yīng)時間為120min。Vero gpELISA和VAR ELISA抗原加保護(hù)劑具有保存穩(wěn)定性和可重復(fù)性。Vero gpELISA檢出WHO VZV國際標(biāo)準(zhǔn)血清的抗體滴度較VAR ELISA提高了4倍,檢出下限分別為195mIU/ml和781mIU/ml,所得結(jié)果與國外成品試劑盒相符。應(yīng)用抗原,通過篩查,獲得了VZV陽性血清,測出的抗VZV IgG幾何平均滴度優(yōu)于人胚肺二倍體2BS細(xì)胞制備的VAR ELISA。 VZV疫苗株在Vero細(xì)胞中能傳代適應(yīng)、制備毒種,用15L瓶旋轉(zhuǎn)培養(yǎng)可大量制備VZV抗原。建立了Vero細(xì)胞制備的VZV IgG VAR ELISA和gpELISA,方法的靈敏度、特異性和可重復(fù)性良好。因此,Vero細(xì)胞用于VZV診斷制品的研究和生產(chǎn)是可行的,本研究為今后兩種試劑盒的標(biāo)準(zhǔn)化和商品化奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R392;R446.6
本文編號：2168434
[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.
【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R392;R446.6
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