口蹄疫病毒VP1、VP4基因核酸疫苗质粒的构建及其免疫原性

Construction and Immunogenicity of DNA Vaccine Plasmid Expressing VP1 and VP4 Genes of Foot-and-Mouth Disease Virus

目的构建口蹄疫病毒(FMDV)VP1、VP4基因核酸疫苗质粒,并研究其免疫原性。方法通过RT-PCR获得FMDVVP1、VP4基因,以真核表达载体pIRESneo为基础,构建VP1单表达以及VP1、VP4融合表达和共表达质粒。转染BHK-21细胞后,Western blot检测目的蛋白的表达。将上述3种基因核酸疫苗、VP1单表达与VP4单表达联合疫苗、灭活疫苗和空载体对照免疫豚鼠,检测血清特异性抗体及中和抗体水平,并进行保护力试验。结果VP1单表达,VP1、VP4融合表达及共表达质粒经PCR及酶切鉴定,证明构建正确。各组核酸疫苗质粒均能诱导产生中和抗体,且具有一定的保护效力。VP1单表达组和联合组保护率均为28.6%;共表达组和融合表达组保护率均为42.9%;灭活疫苗组保护率为100%;空载体组保护率为0。结论口蹄疫病毒VP1与VP4共存时能发挥更好的免疫原性。

Objective To construct the DNA vaccine plasmids expressing the VP1 and VP4 genes of foot-and-mouth disease virus （FMDV）and study its immunogenicity. Methods The VP1 and VP4 genes of FMDV were amplified by RT-PCR, based on which the DNA vaccine plasmids for expression of VP1（pIRESneo-VP1）, fusion expression of VP1 and VP4（pIRESneo-VP1VP4） and coexpression of VP1 and VP4（pIRESeno-VP1-VP4）were constructed and transfected to BHK-21 cells. The expressions of target proteins were identified by Western blot. Guinea pigs were immunized with recombinant plasmids pIRESneo-VP1, pIRESneo-VP1VP4, pIRESneo-VP1-VP4, pIRESneo-VP1 ＋ pIRESneo-VP4, empty vector pIRESneo and inactivated FMDV vaccine respectively, determined for serum specific antibody and neutralizing antibody, then challenged with FMDV type O to evaluate the protection. Results PCR and restriction analysis proved that recombinant plasmids pIRESneo-VP1, pIRESneo-VP1VP4 and pIRESneo-VP1-VP4 were constructed correctly. All the DNA vaccines induced neutralizing antibody and showed a certain protective potency. Both the protective rates of guinea pigs in pIRESneo-VP1 and pIRESneo-VP1 ＋ pIRESneo-VP4 groups were 28. 6%, and those in pIRESneo-VP1VP4 and pIRESneo-VP1-VP4 were 42. 9%, while those in inactivated FMDV and empty vector control groups were 100% and 0 respectively. Conclusion The FMDV containing both VP1 and VP4 showed high immunogenicity as compared with that containing VP1 only.