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目的克隆表达恶性疟原虫复制蛋白PfRPA2亚基,制备多抗,为该蛋白的功能研究奠定基础。方法 PCR扩增目的基因片段,克隆到表达载体pGEX-KG中,构建PfRPA2/pGEX-KG原核表达载体,IPTG诱导表达,SDS-PAGE电泳分析表达产物,谷胱甘肽柱纯化蛋白,Westernblot检测其抗原性,以纯化的GST-PfRPA2免疫小鼠,制备抗PfRPA2的多抗,间接ELISA法检测鼠血清效价,Westernblot鉴定多抗特异性。结果成功构建了重组PfRPA2/pGEX-KG原核表达质粒,并在大肠杆菌中以可溶性形式高效表达,纯化表达产物,制备抗PfRPA2的鼠多抗,效价为10-7,Westernblot证实此抗体可识别恶性疟原虫内与PfRPA2蛋白位置对应的特异性条带。结论恶性疟原虫复制蛋白PfRPA2亚基在大肠杆菌中获得可溶性高效表达,纯化表达产物能诱导小鼠生产能识别天然蛋白的特异性抗体。
Objective To clone and express the PfRPA2 subunit of Plasmodium falciparum, and to prepare polyclonal antibodies, which laid the foundation for the function of this protein. Methods The target gene fragment was amplified by PCR and cloned into the expression vector pGEX-KG. The prokaryotic expression vector PfRPA2 / pGEX-KG was constructed and induced by IPTG. The expressed product was analyzed by SDS-PAGE and purified by glutathione. The protein was detected by Western blot Antigens were used to immunize mice with the purified GST-PfRPA2 to prepare polyclonal antibodies against PfRPA2. The serum titer was determined by indirect ELISA and the specificity of polyclonal antibody was identified by Western blot. Results The prokaryotic expression plasmid pfRPA2 / pGEX-KG was successfully constructed and expressed in soluble form in E. coli. The expressed product was purified and the polyclonal antibody against PfRPA2 was prepared with titer of 10-7. Western blot confirmed that this antibody can recognize Specific bands corresponding to the PfRPA2 protein in Plasmodium falciparum. Conclusion The PfRPA2 subunit of Plasmodium falciparum was soluble and highly expressed in Escherichia coli. The purified product could induce the production of specific antibodies that recognize natural proteins in mice.