恶性疟原虫重组复合抗原的分离纯化及免疫活性鉴定

THE PURIFICATION AND IMMUNOLOGICAL CHARACTERIZATION OF RECOMBINANT HYBRID ANTIGEN OF PLASMODIUM FALCIPARUM

高效表达恶性疟原虫保护性抗原重组复合蛋白的工程菌ｐＷＲ－Ｃ／ＪＭ１０９和ｐＷＲ－ＣＡＣ／ＪＭ１０９，在发酵罐中发酵后收集菌体，经超声波破菌，硫酸铵分级沉淀，分子筛和离子交换层析等步骤纯化后，纯度可达８０％以上。纯化后的融会蛋白具有β－半乳糖苷酶的活力，用等电聚焦技术测定纯化蛋白的等电点分别为８．４和８．２５。用Ｄｏｔ－ＥＬＩＳＡ法测定纯化蛋白的免疫原性，证实纯化的重组蛋白可与小鼠抗恶性疟原虫抗体以及疟区病人血清发生特异性免疫反应；重组蛋白免疫家兔后制备的抗血清可特异性地识别恶性疟原虫海南株和云南株的红内期抗原，说明我们纯化的融合蛋白为具有恶性疟原虫抗原活性的重组复合抗原。

The recombinant bacterial strains pWR-C/JM109 and pWR-CAC/JM109 whichexpress hybrid protective antigens of P.falciparum were cultivated and induced in fermentationtank. the bacteria were harvested and broken by ultrasonication. The expressed proteins werepartially purified by ammunium sulfate fractionation, gel filtration and ion-exchange chromatog-raphy reaching a purity of 82%. the purified fused proteins show the activities of β-galactosidase.The iso-electric points of the fused proteins are 8.4 and 8. 25 tested by isoelectronic focusing.The immunogenicity of hybrid protein was tested by dot-ELISA. It was shown that purified re-combinant protein can specifically react with mouse antibodies against P.falciparum and the fal-ciparum patient serum from Yunnan Province. The proteins were used to immunize rabbit to pre-pare antibodies and the immune serum can specifically recognize the blood stage antigens ofHainan and Yunnan strains of Palciparum. This indicates that the purified protein is the anti-genicity of P.falciparum antigens.