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目的恶性疟原虫组氨酸富集蛋白Ⅱ（Ｈｉｓｔｉｄｉｎｅ－ｒｉｃｈｐｒｏｔｅｉｎⅡ，ＨＲＰⅡ）是疟原虫红内期生活史过程中从感染红细胞分泌至血浆中的水溶性抗原，是重要的诊断用抗原。本文通过实现恶性疟原虫ＨＲＰⅡ在大肠杆菌中的表达，为研制用于疟疾诊断的抗ＨＲＰⅡ单克隆抗体奠定基础。方法将含有ＨＲＰⅡ抗原基因的重组表达质粒ＨＲＰⅡ／ｐＥＴ８ｃ用钙法转化大肠杆菌ＢＬ２１（ＤＥ３），重组子经聚合酶链反应（ＰＣＲ）和ＢａｍＨＩ与ＢｇｌⅡ双酶切鉴定。重组子ＨＲＰⅡ／ｐＥＴ８ｃ／ＢＬ２１（ＤＥ３）在２８℃条件下，用１ｍＭＩＰＴＧ诱导表达，ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ免疫印迹分析表达产物。结果成功构建ＨＲＰⅡ／ｐＥＴ８ｃ重组质粒，在低温条件下经ＩＰＴＧ诱导，ＨＲＰⅡ呈可溶性、非融合性表达，ＳＤＳ－ＰＡＧＥ分析表明表达产物的分子量约３３ｋＤａ，薄层扫描显示表达蛋白占菌体总蛋白的２０．７６％。Ｗｅｓｔｅｒｎ免疫印迹表明，表达产物能被抗ＨＲＰⅡ人工合成九肽单克隆抗体特异识别。结论恶性疟原虫ＨＲＰⅡ在原核表达系统ｐＥＴ８ｃ／ＢＬ２１（ＤＥ３）中获得成功表达，为基因工程大规模制备生产ＨＲＰⅡ抗原奠定基础
Objective Histidine-rich protein Ⅱ (HRPⅡ) is an important diagnostic antigen for Plasmodium falciparum (HRPⅡ), a water-soluble antigen secreted from infected erythrocytes into plasma during the erythrocytic red blood cell life cycle. In this paper, the expression of Plasmodium falciparum HRPII in Escherichia coli was established, which laid the foundation for the development of anti-HRPII monoclonal antibody for diagnosis of malaria. Methods Recombinant plasmid HRPⅡ / pET8c containing HRP Ⅱ antigen was transformed into E. coli BL21 (DE3) by calcium method. The recombinant was identified by double enzyme digestion with BamHI and Bgl Ⅱ. The recombinant HRPⅡ / pET8c / BL21 (DE3) was induced by 1mM IPTG at 28 ℃, and the expressed product was analyzed by SDS-PAGE and Western immunoblotting. Results Recombinant plasmids of HRPⅡ / pET8c were successfully constructed and induced by IPTG at low temperature. HRPⅡ was soluble and non-fusion, SDS-PAGE analysis showed that the molecular weight of the product was about 33kDa. The results of TLC showed that the expressed protein accounted for 20.76%. Western immunoblotting showed that the expressed product was specifically recognized by anti-HRPII synthetic nonapeptide monoclonal antibody. Conclusion Plasmodium falciparum HRPⅡ was successfully expressed in prokaryotic expression system pET8c / BL21 (DE3), which laid the foundation for gene engineering large-scale production of HRPⅡ antigen