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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 polyclonal antibody by using Escherichia coli to express PF3D7_1361800 protein. Methods According to the water-soluble analysis of PF3D7_1361800 protein, the target gene primers were designed, and the target gene fragment was amplified by PCR and the length was 897 bp. The fragment was cloned into pMD18-T vector and ligated into pET-28a and pGEX-4T-1 expression vector. After sequencing, the recombinant plasmid was transformed into Escherichia coli BL21-CodonPlus (DE3) RIPL, the expression was induced with 0.1 mmol / L IPTG, and the target protein was purified with His GraviTrapTM and Gluthathione-Sepharose 4B affinity chromatography. Rabbits were immunized with His-tag recombinant protein and immunized once every two weeks. Blood was collected at 14, 28, 42 and 52 days after immunization, respectively. The antibody titer was measured by indirect ELISA. The prepared polyclonal antibody was used as a primary antibody and its specificity was analyzed by Western blot. Results After sequencing and restriction enzyme digestion, the expression vectors pET-PF3D7_1361800 and pGEX-PF3D7_1361800 were successfully constructed. SDS-PAGE analysis of affinity purification of the recombinant protein purity better, indirect ELISA method to detect the protein immune rabbit serum antibody titers> 1:16 000. Conclusion The PF3D7_1361800 expression vector was successfully cloned and the target protein was expressed. The prepared anti-recombinant protein polyclonal antibody is specific, which lays the foundation for the study of the biological function of the protein and the development of the malaria vaccine.