马巴贝西虫EMA-1基因的克隆与表达

Cloning and Expression of EMA-1 Gene of Babesia equi

通过聚合酶链式反应扩增出体外培养的马巴西虫USDA株基因EMA_1 ,将该片段连接到克隆质粒载体pUC1 9的BamHI酶切位点上 ,并对其序列进行测定。结果表明该基因长度为 83 6bp,编码的表面蛋白由 2 72个氨基酸残基组成 ,与佛罗里达 (Florida)株的EMA_1的氨基酸序列有 95 %左右的同源性。再将EMA_1基因片段插入到表达质粒载体pGE MEX_2的BamHI位点上 ,并导入大肠杆菌JM10 9(DE3 )中。经SDSPAGE分析和Westernblot检测的结果表明 ,马巴贝西虫USDA株基因EMA_1 ,在大肠杆菌内获得表达 ,融合蛋白的分子量为 65kDa。将其免疫给小鼠可产生特异性的抗马巴贝西虫抗体 ,且同驽巴贝西虫无交叉反应。

EMA-1 gene of B epui USDA strain in vitro was amplified by polymerase chain reaction method,then the PCR product was ligated into BamHI site of transfer vector of pUC19,and was sequenced.Results showed that there are 836bp in EMA-1 gene,which encode 272 amino acid residues of surface protein.Copmared sequence to sequence of EMA-1 gene of Florida stains,there is 95% homology.The EMA-1 gene was inserted into BamHI site of expressing vactor pEGMEX-Z,and expressin E.coli JM 109(DE3)strain.The EMA-1 protein of B.equin USDA strain expressed in E.coli was edtected by SDS-PAGE and Western blot and the molecular weight of fusion protein is 65Kda.Mice immunized with EMA-1 could produce specific antibody anaginst B.equi and there was't cross reactions with B.Caballi.