【作者】 张赟硕；

【导师】 李一经；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 分别用昆虫细胞杆状病毒表达系统表达的重组猪传染性胃肠炎病毒(Transmissible gastroenteritis virus，TGEV)S蛋白和含有猪传染性胃肠炎病毒TH-98株S基因主要抗原位点的重组质粒PCI-Sa免疫BALB／c小鼠，按常规方法进行细胞融合。间接ELISA方法进行筛选，前者筛选抗原为TGEV细胞培养物上清；后者筛选抗原为纯化的乳酸乳球菌表达重组TGEV S蛋白。采用有限稀释法，经过三次克隆，最终前者获得两株抗TGEV S蛋白的杂交瘤细胞株(C7C882和B5G8G7)；后者获得一株抗TGEV S蛋白的杂交瘤细胞株(E6D9A12)。三株杂交瘤细胞分泌的单克隆抗体亚类分别为IgM、IgM、IgG2a类，杂交瘤细胞体外长期培养和冻存均不影响抗体的分泌。杂交瘤细胞的平均染色体数为88±10对，用相应的筛选抗原经间接ELISA检测C7C882、B5G8G7、E6D9A12细胞培养上清的抗体效价分别为1：200、1：100、1：500，腹水抗体效价分别为1：2×10~5、1：1×10~4、1：6×10~5。以猪传染性胃肠炎病毒(TGEV)、猪流行性腹泻病毒(PEDV)、猪轮状病毒(PRV)、猪伪狂犬病病毒(PrV)细胞培养物上清液为抗原，与三株杂交瘤细胞上清经间接ELISA检测结果显示，这三株单抗与TGEV显示良好的特异性，而与PEDV、PRV和PrV不存在交叉反应。说明获得的单抗具有高度的特异性。用实验获得的单克隆抗体和SP2／0细胞培养上清对感染TGEV的ST细胞培养物进行了间接免疫荧光检测病原的比较研究：间接免疫荧光染色后，与单抗作用的细胞培养物中，多数细胞在其胞浆和细胞膜上出现黄绿色闪亮荧光，且荧光强度强，与SP2／0细胞培养上清作用的细胞培养物未见有黄绿色闪亮荧光，结果表明，利用所制备的抗TGEV S蛋白的单克隆抗体进行间接免疫荧光对病原检测具有较高的特异性。以纯化的TGEV为抗原，Dot-ELISA检测实验获得的单克隆抗体与TGEV的反应性，结果三株杂交瘤细胞上清与TGEV反应均出现灰色印记，可判定为阳性；与SP2／0细胞上清反应为阴性。证明单克隆抗体与TGEV具有特异性反应。上述实验结果均表明，所制备的抗TGEV S蛋白的单克隆抗体对病原检测具有较强的特异性，为TGEV病原特性的基础性研究和病原的快速检测提供了重要的物质基础。更多还原

【Abstract】 In this research, recombinant spike protein of transmissible gastroenteritis virus(TGEV) whichwas expressed with baculovirus expression system and a recombinant plasmid named PCI-Sa withprimary epitopes of TGEV were used to immune BALB/c mouses respectively. Cell fusion wasperformed according to the traditional method. Indirect ELISA assay was carried out to screenhybridoma cell lines using supernatant of TGEV propagation as for the first kind of antigen, andpurified recombinant spike protein expressed in Lactococcus lactics for the second kind of antigen.By limiting dilution and 3 serials of clones, 2 hybridoma cell lines (designated C7C8B2 andB5G8G7) producing antibodies against TGEV were obtained, and 1 hybridoma cell line(designated E6D9A12) secreting antibodies recognizing S protein was also gained. Theantibodies of 3 hybridoma cell lines were respective belong to IgM、IgM、IgG2a subgroup. Afterextended culture and several freeze thaw cycles, monoclonal antibodies could still be stablysecreted. The average number of chromosomes in 3 hybridomas was 88±10 couples. Investigatedthrough indirect ELISA, the titers of 3 cell-cultured antibodies were measured to be 1:200, 1:100,1:500 and titers of antibodies produced in ascities were respectively 1:2×10~5, 1:1×10~4, 1:6×10~5.Using the supernatants of TGEV, PEDV, PRV and PrV propagation as antigens, 3 antibodiesall exposed specific recognition characters. And they could not react with PEDV, PRV or PrV. Itwas confirmed that these specific antibodies could be used for TGEV diagnosis.Immunofluorescence assays were performed with ST cell monolayers infected with TGEVprobed by the antibodies or supernatant of SP2/0 cell culture. After staining with FITC labeledsecondary antibodies, strong yellow-green fluorescence could be observed in plasma and onmembrane of cells. However, no fluorescence was detected with the addition of supernatant ofSP2/0 cell culture. With recombinant S protein as immunogen, antibodies were found tospecifically bind to pathogens with the confirmation of indirect immunofluorescence assays.Using purified TGEV as antigen, dot-ELISA was performed to determine the correlationbetween the monoclonal antibodies and TGEV. It was revealed that the supernatants of 3hybridomas but not supernatant of SP2/0 cell culture could recognize TGEV. The specificity ofantibodies to virus was further ensured.All the results indicate that with recombinant S protein as immunogen, the screenedmonoclonal antibodies own rather excellent specificity to TGEV. And this could provide foundationfor TGEV pathogenesis research and rapid diagnosis.更多还原