Asia-Ⅰ口蹄疫病毒P1-2A基因真核表达质粒的构建及小鼠免疫试验

目的 构建含有Asia-Ⅰ型口蹄疫病毒(FMDV)衣壳蛋白前体P1-2A 基因的真核表达质粒pVAXⅠ-P1-2A，并用pVAXⅠ-P1-2A免疫小鼠，评价其体液免疫和细胞免疫水平。方法 通过RT-PCR 方法扩增得到含有FMDV P1-2A编码区的目的基因，并将其克隆到pMD18-T载体上。将pMD18-T-P1-2A和 pVAXⅠ分别经EcoRⅤ和XbaⅠ双酶切后连接构建真核表达质粒pVAXⅠ-P1-2A。将酶切鉴定正确后的重组质粒转染HeLa细胞进行IFA检测。再进行小鼠血清特异性抗体试验、小鼠T淋巴细胞增殖试验和IFN-γ ELISPOT试验。结果 酶切结果与预期目的条带大小相符；荧光结果表明经pVAXⅠ-P1-2A转染的细胞有明显的黄绿色荧光，说明P1-2A基因在HeLa细胞中得到了表达；小鼠免疫结果表明，用免疫的小鼠都产生了较强的体液免疫和细胞免疫，并且T淋巴细胞增殖数和产生IFN-γ的细胞数和对照组相比显著提高(P<0.05)。间接ELISA试验表明，在免疫第14天抗体水平比对照组明显增高(P<0.05)。结论 成功构建了真核表达质粒pVAXⅠ-P1-2A,并通过小鼠免疫试验发现重组质粒试验组能够诱导产生特异性的体液免疫和细胞免疫应答。

Construct of the plasmids of Asia-Ⅰ FMDV P1-2A genes expressing in eukaryotic and immunnity test

Objective Construct eukaryotic expression plasmid pVAXⅠ- P1-2A containing capsid protein precursor P1-2A gene of Asia-ⅠFMDV ,and evaluate cellular and humoral immunity level after immunization mice with pVAXⅠ- P1-2A. Methods We amplied the coding region of FMDV P1-2A by RT-PCR and inserted it to Vector pMD18-T. Eukaryotic expression plasmid pVAXⅠ- P1-2A was constructed by connecting pMD18-T-P1-2A and pVAXⅠdigested by double restriction endonuclease EcoRⅤand XbaⅠ. We transfected the right recombinant plasmid confirmed by endonuclease digestion into the Hela cell ,and perform IFA to assure the right expresstion. After this, mice serological specific antibody test ,T lymphocyte proliferation assay and IFN-γ ELISPOT test were carried out. Results The result of restriction endonuclease digestion was accordance with the anticipated objective strap size . IFA showed that there are flavovirens fluorescence in cells transfected with pVAXⅠ-P1-2A ,and confirmed the expression of P1-2A gene in the HeLa cell; The result of mice immunization test indicated that there are stronger cellular and humoral immunity response , and more T lymphocyte and the cells that produce IFN-γin immunized mice than the control groups (P<0.05). The indirect ELISA test manifested that the antibody level was higher remarkably than the control groups (P<0.05) in the 14th day after immunity. Conclusion we have successfully constructed eukaryotic expression plasmid pVAXⅠ- P1-2A ,and confirmed that specific humoral immunity and cellullar immunologic response can be induced in the group injected with recombinant plasmids with mice immunization test.