恶性疟原虫3D7株新型裂殖子蛋白质(PF3D7_1361800)的表达纯化及多克隆抗体的制备

Expression and purification of the new merozoite protein PF3D7_1361800 and preparation of polyclonal antibodies

目的用大肠埃希菌表达PF3D7_1361800蛋白,制备多克隆抗体。方法根据PF3D7_1361800蛋白的水溶性分析,设计目的基因引物,采用PCR技术扩增目的基因片段,长度为897bp。将其克隆到pMD18-T载体,测序正确后将该片段连接到pET-28a和pGEX-4T-1表达载体中,测序鉴定正确后将重组质粒转化到大肠埃希菌BL21-CodonPlus(DE3)-RIPL中,用0.1mmol/L IPTG诱导表达,用His GraviTrapTM和Gluthathione-Sepharose 4B亲和层析柱纯化目的蛋白质。用His-tag重组蛋白免疫家兔,每2周免疫一次,分别于免疫后14、28、42、52d采血,用间接ELISA法测定抗体效价。以制备的多克隆抗体作为一抗,采用Western blot分析其特异性。结果经测序和酶切鉴定,成功构建了表达载体pET-PF3D7_1361800和pGEX-PF3D7_1361800。SDS-PAGE分析亲和纯化的重组蛋白纯度较好,间接ELISA法检测该蛋白免疫兔血清抗体效价>1∶16 000。结论成功克隆了PF3D7_1361800的表达载体并表达目的蛋白,制备的抗重组蛋白多克隆抗体具有特异性,为研究该蛋白质的生物学功能及疟疾疫苗的研发奠定了基础。

Objective To express the protein PF3D7_1361800in E.coli and prepare polyclonal antibodies. MethodsBased on analysis of the water solubility of the protein PF3D7_1361800,apair of primers was designed,and fragments amplified by PCR were about 897 bp in length.PCR products were cloned into pMD18-T,and the correct fragment was ligated into pET-28 aand pGEX-4T-1expression vectors after sequencing.These recombinant plasmids were transferred into BL21-CodonPlus（DE3）-RIPL E.coli.The recombinant protein was expressed after induction with 0.1 mmol/L IPTG and the protein was then purified with His GraviTrapTMand Gluthathione-Sepharose 4B.Polyclonal antibodies against PF3D7_1361800were generated in rabbits immunized with purified His-tagged recombinant protein.Each rabbit was immunized every two weeks.A blood sample was collected after 14 d,28d,42 d,and 52 d.The polyclonal antibody titer was detected with indirect ELISA assay.The polyclonal antibody served as the primary antibody,and Western blotting was used to detect its specificity. Results The expression vectors pET-PF3D7_1361800and pGEX-PF3D7_1361800were successfully constructed and indicated by sequencing and restriction enzyme digestion.Results of SDSPAGE indicated that the proteins were highly pure.Indirect ELISA indicated the serum titer of antibodies to PF3D7_1361800was higher than 1︰16 000.Western blotting indicated that the polyclonal antibody was highly specific to the natural protein. Conclusion The PF3D7_1361800gene was successfully cloned and expressed in this study,and polyclonal antibodies were then obtained.These antibodies were specific,establishing a foundation for the further study of the biological function of this protein as well as the development of related malaria vaccines.