恶性疟原虫复合多表位基因的原核表达和多克隆抗体制备

原核表达恶性疟原虫多期多表位基因并制备其多克隆抗体。在前期构建恶性疟原虫复合多期多表位重组真核表达载体的基础上,将测序正确的AMAMEG基因克隆入原核表达载体pGEX4T—1并诱导表达。采用包涵体洗涤的方式纯化目的蛋白,以AMA—1抗体对纯化蛋白进行Western—blot分析。将纯化的融合蛋白免疫小鼠制备多克隆抗体。通过PCR成功获得长度为1 000 bp的恶性疟原虫AMA—1胞外域基因片段,获得了与多表位基因连接后的AMAMEG基因。通过诱导表达,显示在相对分子量约95 000处有预计大小的特异性条带,表明成功表达出融合蛋白。经此纯化的融合蛋白免疫的小鼠能产生特异性抗体,其滴度达到1∶105。纯化的融合蛋白和多克隆抗体为研究新型疟疾疫苗奠定一定的理论和实践基础。

Expression and Preparing Antibody of Fusion Multiepitope Gene of Plasmodium falciparum

To express and purify Plasmodium falciparum fusion multiepitope gene in E.coli and to prepare polyclonal antibody,with the plasmid containing the gene coding AMA—1 of P.f.YN and the gene coding cryptic epitopes by amino modified acid substitution were inserted into pGEX4T—1,obtained the recombinant plasmid pGEX4T—1/AMAMEG.The recombinant vector was transformed into BL21(DE3).After induction,induced fusion protein was purified by purified inclusion bodies washing.The purified protein was injected into the Balb/C mice.And the titer of the mice’s anti-serum was measured by ELISA.The results show that recombinant plasmid pGEX4T—1/ AMAMEG coding multi-CTL epitope gene and AMA—1 gene is obtained successfully.The GST/AMAMEG fusion protein is successfully purified and the anti-serum with high titer is obtained.The preparation of GST/AMAMEG fusion protein and polyclonal antibody can be used for further investigation.