摘要
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FMDVP1-2A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,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。
Objective： The eukaryotic expression plasmid pVAX Ⅰ- P1-2A containing capsid protein precursor P1-2A gene of Asia- Ⅰ FMDV was constructed. Cellular and humoral immunity level of the mice immunized against pVAX Ⅰ - P1-2A was evaluated. Methods：The gene of FMDV P1-2A was amplified by RT- PCR, inserted into vector pMD18-T, then subcloned to eukaryotic expression vector pVAX Ⅰ. The eukaryotic expression plasmid pVAX Ⅰ- P1-2A transfected into the Hela cell, and detected by IFA. The mice were immunized against pVAX Ⅰ- P1-2A, the FMDV specific antibody was detected by ELISA, T lymphocyte proliferation analyzed by MTT, IFN-γ was detected by ELISPOT method. 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 pVAX Ⅰ -P1-2A, and confirmed the expression of P1-2A gene in HeLa cell. 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, and more T lymphocyte and the cells that produce IFN-γ in immunized mice than the control groups （ P 〈 0.05 ）. The result showed that there are significant cellular and humoral immunity response in the mice immunized with pVAX Ⅰ- P1-2A. Conclusion： Eukaryotic expression plasmid pVAX Ⅰ - P1-2A was successfully constructed, and pVAX Ⅰ - P1-2A could induce specific humoral and cellullar immunity response in mice.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2008年第8期31-35,共5页
China Biotechnology
基金
国家“863”计划(2006AA10A204)资助项目
