恶性疟原虫海南分离株FCC1/HN有性期特异抗原Pfs48/45基因的克隆与表达

CLONING AND EXPRESSION OF THE GENES ENCODING FOR THESEXUAL STAGESPECIFIC ANTIGENS OF PLASMODIUMFALCIPARUM,SOUTHERN CHINA ISOLATE FCC1/HN*

采用基因工程技术，将编码恶性疟原虫有性期特异抗原Ｐｆｓ８／４５的基因克隆到真核表达质粒ｐｃＤ－ＮＡ３，并进行ＤＮＡ序列测定，再通过磷酸钙—ＤＮＡ共沉淀转化法将重组质粒ｐｃＤＮＡ３－ｐＦＳ４８／４５导入ＨｅＬａ细胞，建立稳定分泌Ｐｆｓ４８／４５蛋白的阳性克隆株。结果显示，我国海南ＦＣＣ１／ＨＮ株Ｐｆｓ４８／４５抗原基因序列与ＮＦ５４株者高度同源，提示该基因在不同虫株间高度保守，是研制疟疾疫苗的理想靶抗原；在ＨｅＬａ细胞中表达的Ｐｆｓ４８／４５蛋白分子量约为４６／４３．５ｋＤａ双联体蛋白，其表达量占细胞培养上清蛋白总量的１８．２７％。经ＷｅｓｔｅｒｎＢｌｏｔ分析显示，表在蛋白能被配子体免疫鼠血清特异性识别，提示表达的重组蛋白Ｐｆｓ４８／４５具有免疫活性。真核表达系统ｐｃＤＮＡ３／Ｐｆｓ４８／４５／ＨｅＬａ的建立为进一步研究重组Ｐｆｓ４８／４５抗原的免疫原性和保护性奠定基础。

Using genetic engineering techniques ,the gene encoding the gametocyte/ gametespecific membrane protein Pfs48/45 of Plasmodium falciparum isolate FCC1/HN was cloned into eukaryotic expression plasmid pcDNA3,then sequenced by the dideoxynucleotide terminator method with 5end primer P1 and compared with NF54 isolate (African origin).Employing the calcium phosphateDNA coprecipitation technique,the recombinant plasmid pcDNA3Pfs48/45 was transformed into mammalian cell line of human HeLa cells,to establish HeLa cell line expressing Pfs48/45 antigen.The results showed that the nucleotide sequence of Pfs48/45 of Plasmodium falciparum isolate FCC1/HN from Hainan,southern China was highly homologus with that of the isolate NF54,which proved that the gene encoding the sexual stagespecific antigens of Pfs48/45 is highly conserved at different isolates of Plasmodium falciparum,suggesting that Pfs48/45 is an important target antigen for the development of protective malaria vaccine.The molecular weight of the expressed protein Pfs48/45 is about 46 and 435kDa as a doublet 46/435 proteins,similar to the predicted value.The expressed amount of recombinant Pfs48/45 was up to 1827% of the total proteins in the supernatants of cultures.In addition,the expressed protein could be specifically recognized by BALB/c Mouse antigametocyte serum,suggesting that the expressed products exhibit the immunological activity of Pfs48/45 of Plasmodium falciparum.The further studies on the immunogenicity and protective immunity of the recombinant protein Pfs48/45 are under investigation.