盖他病毒Cap和E2蛋白多克隆抗体的制备

Preparation of Polyclonal Antibodies Against Cap and E2 Proteins of Getah Virus

为了制备盖他病毒(Getah virus,GETV)结构蛋白Cap和E2的多克隆抗体,将Cap和E2克隆到原核表达载体pET-32a中,然后将构建的表达质粒pET-32a-Cap/E2转化大肠埃希氏菌BL21(DE3)菌株,用IPTG诱导蛋白表达,非变性条件下用Ni-NTA琼脂糖树脂纯化重组pET-32a-Cap/E2蛋白。用纯化的重组蛋白多次接种新西兰大白兔,从而制备多克隆抗体,后续进行效价测定、Western blot与间接免疫荧光试验(IFA)验证抗体与病毒蛋白的反应。结果表明,Western blot与IFA显示制备的兔多克隆抗体能够与GETV抗原特异性结合;效价测定表明了制备的兔多克隆抗体Cap和E2效价均可达到1∶64000。研究制备的多克隆抗体特异性良好,为GETV检测制剂的研发提供了良好的生物材料,为GETV蛋白功能的研究奠定了基础。

To prepare polyclonal antibodies against the structural proteins Cap and E2 of Getah virus(GETV),the recombinant expression plasmids pET-32a-Cap/E2 were constructed and transformed into E.coli BL21(DE3)strain.The prokaryotic expression of recombinant Cap/E2 proteins in BL21(DE3)was induced by isopropylβ-D-thiogalactopyranoside(IPTG)and then purified by Ni-NTA agarose resin under non-denaturing conditions.Polyclonal antibodies were produced by subcutaneous injection of New Zealand white rabbits with the purified recombinant proteins.Titration,indirect immunofluorescence and Western blot were performed to test the reaction of antibodies with virus proteins.The results of Western blot and IFA showed that the polyclonal antibodies could bind specifically to the antigen of GETV,and the titer of polyclonal antibodies was as high as 1:64000.The polyclonal antibodies prepared in this study provided a good biological material for diagnostic reagent development for GETV and laid a solid foundation for further research on function of GETV structural proteins.