猪伪狂犬病病毒gB、gC和gD蛋白的表达及诊断抗原筛选

Expresson of gB,gC and gD Proteins of Pseudorabies Virus and Screening as Diagnostic Antigens

为研发猪伪狂犬病的免疫学诊断试剂,利用杆状病毒表达系统表达了猪伪狂犬病病毒(PRV)gB、gC和gD蛋白,并对蛋白进行SDS-PAGE和Western blot鉴定;将纯化后的3种蛋白分别制备成不同蛋白类型的疫苗免疫健康仔猪,比较蛋白的免疫原性;将3种蛋白分别作为包被抗原建立间接ELISA方法,检测从不同地区收集的临床血清,与PRV中和试验检测结果进行了比较。结果显示,成功拯救出重组杆状病毒rPRV-gB-His株、rPRV-gC-His株和rPRV-gD-His株,经IFA鉴定,均可与猪伪狂犬病病毒阳性血清发生特异性反应;表达出的蛋白经SDS-PAGE和Western blot鉴定,可见大小分别约为120 ku、65 ku和50 ku的蛋白条带;3种蛋白免疫原性比较结果显示gD蛋白疫苗免疫组猪血清中和效价最高(1∶22.4~1∶32.0);3种蛋白建立的间接ELISA方法检测30份临床血清,结果gD蛋白作为包被抗原建立的ELISA方法与PRV中和试验的符合率最高(100.0%)。表明gD蛋白是更好的诊断抗原,可用于PRV抗体检测试剂盒的开发。

In order to develop pseudorabies immunodiagnostic reagents,PRV gB,gC,and gD proteins were expressed in baculovirus,and the proteins were identified by SDS-PAGE and Western blot.In order to compare the immunogenicity of the proteins,three purified proteins were prepared into vaccines respectively to immunize healthy piglets.Indirect ELISA were established with the three proteins respectively as coating antigen and were used to detect clinical sera collected from different areas,the results were compared with PRV neutralization test.The results showed that the recombinant baculovirus rPRV-gB-His,rPRV-gC-His and rPRV-gD-His strains were rescued successfully,according to IFA identification,the three recombinant baculovirus strains could have specific reaction with positive serum of pseudorabies.SDS-PAGE and Western blot identification showed that the expressed proteins were 120 ku,65 ku and 50 ku respectively.The immunogenicity comparison of the proteins showed that neutralization titer was highest(1∶22.4-1∶32.0)in pigs immunized with gD protein vaccine.Thirty clinical sera were detected by indirect ELISA established with three proteins,the results showed that the ELISA method established with gD protein had the highest coincidence rate with PRV neutralization test(100.0%).These results indicated that gD protein was a better diagnostic antigen and could be used for the establishment of kits for detecting antibody against PRV.