猪繁殖与呼吸综合征病毒GP5、M蛋白基因核酸疫苗表达质粒的构建及其免疫原性

Construction and Immunogenicity of Recombinant Expression Vectors for GP5 and M Proteins of Porcine Reproductive and Respiratory Syndrome Virus

目的构建猪繁殖与呼吸综合征病毒(PRRSV)美洲株GP5、M蛋白基因核酸疫苗表达质粒,并检测其免疫原性。方法用RT-PCR法扩增PRRSV的GP5和M蛋白基因片段,克隆至真核表达载体pIRES-neo中,构建真核表达质粒pIRES-G和pIRES-M,制备核酸疫苗,以其单独或联合免疫小鼠,检测其免疫原性。结果真核表达质粒pIRES-G和pIRES-M经酶切鉴定证明构建正确。第1次免疫后4周,各重组质粒免疫组小鼠血清ELISA抗体水平均显著升高,其中pIRES-G + pIRES-M组抗体水平最高;第1次免疫后9周,pIRES-G + pIRES-M组小鼠血清中和抗体效价(1∶16)高于pIRES-G组(1∶8)和pIRES-M组(1∶4),但均低于灭活疫苗组(1∶64);第1次免疫后9周,与pIRES-neo组相比,各组免疫小鼠脾细胞经特异性(PRRSV)和非特异性(ConA)抗原刺激后,均有明显的增殖,对非特异性刺激反应比特异性刺激反应略强。结论已成功构建PRRSV GP5、M蛋白基因核酸疫苗表达质粒,二者联合免疫效果更好。

Objective To construct the recombinant expression vectors for GP5 and M proteins of porcine reproductive and respiratory syndrome virus （PRRSV）, as a DNA vaccine, and determine its immunogenicity. Methods The GP5 and M gene fragments of PRRSV were amplified by RT-PCR and cloned into eukaryotic expression vector pIRES-neo to construct recombinant plasmids pIRES-G and pIRES-M respectively. Mice were divided into 3 test and 2 control groups. The mice in 3 test groups were immunized for 3 times with recombinant plasmids pIRES-G, pIRES-M and pIRES-G ＋ pIRES-M, while those in 2 control groups with plusmid pIRES-neo and a domestic inactivated PRRSV vaccine, respectively. The ELISA antibody and neutralizing antibody in sera as well as splenic lymphocyte proliferation level were determined before and at various weeks after immunization. Results Restriction analysis proved that recombinant plasmids pIRES-G and pIRES-M were constructed correctly. The ELISA antibody titcrs in sera of mice 4 weeks after the 1st immunization increased significantly in pIRES-G, pIRES-M and pIRES-G ＋ pIRES-M groups, which was significantly higher in pIRES-G ＋ pIRES-M group than in the other 2 test groups. Nine weeks after the 1st immunization, the neutralizing antibody titers in sera of mice in pIRES-G ＋ pIRES-M group （1 ： 16） was higher than those in pIRES-G （1 ： 8） and pIRES-M （ 1 ： 4） groups, while lower than those in inactivated PRRSV vaccine group （ 1 ： 64）. Meanwhile, compared with those in pIRES-neo group, the splenic lymphocytes of mice in pIRES-G ＋ pIRES-M, pIRES-G, pIRES-M and inactivated PRRSV vaccine groups proliferated significantly after stimulation with specific＇ （PRRSV） or non-specific （ConA） antigen. However, the proliferation level of splenic lymphocytes after stimulation with ConA was slightly higher than that with PRRSV antigen. Conclusion The recombinant expression vectors for GP5 and M proteins of PRRSV was successfully constructed, and the combined immunization with them showed satisfactory immune effect.