伊氏锥虫HGPRT基因cDNA的克隆及其在E.coli中的表达  

Cloning and Expression of HGPRT cDNA of Trypanosoma evansi

作　　者：刘全[1] 王祥生[1] 

机构地区：[1]解放军军需大学军事兽医研究所,长春130062

出　　处：《畜牧兽医学报》2003年第6期597-599,共3页ACTA VETERINARIA ET ZOOTECHNICA SINICA

摘　　要：根据引物设计的一般原则,参照伊氏锥虫HGPRT基因的核苷酸序列设计、合成一对引物,PCR扩增伊氏锥虫HGPRT基因cDNA。低熔点琼脂糖回收PCR产物,并将其克隆至pGEM-TEasy载体中,经酶切、PCR鉴定和序列分析,获得重组中间质粒pGEM/HGPRT。重组中间质粒pGEM/HGPRT经NcoⅠ和SalⅠ双酶切,回收目的片段HGPRT,以非融合形式插入原核表达质粒pBV220构建表达质粒pBV/HGPRT,转化大肠杆菌DH5α,42℃诱导表达,聚丙烯酰胺凝胶电泳和薄层扫描分析,表达产物HGPRT的分子量约为23kD,与理论推算值相符,表达率占总菌体蛋白的19%,经间接ELISA检测,表达产物能被伊氏锥虫阳性血清所识别。A pair of PCR primer was designed referring to the HGPRT gene of Trypanosoma evansi and PCR was proceeded to get the HGPRT gene of T. evansi.The result that there was a bright band between 500bp and 750bp of DL2000 Marker conformed to theoretical value.The PCR product was regained by the low melting point agarose and cloned into pGEMT Easy vector.The plasmid,pGEM/HGPRT was constructed by inserting the HGPRT cDNA into pGEMT Easy vector.By analysis of restriction endonucleases,PCR and nucleotide sequencing,the plasmid contained HGPRT cDNA.The target gene was recovered from the recombinant of pGEM/HGPRT and subcloned into expression vector pBV220,resulting in recombinant pBV/HGPRT,and transformed E.coli DH5α.It was verified that the HGPRT cDNA have integrated in the proper position and correct orientation with the methods of restriction endonucleases digestion and PCR.The host cells,containing pBV/HGPRT were induced by 42℃ for 4h.Analysed by SDSPAGE,the target protein was shown to be about 23kD,similar to the putative MW,SDSPAGE intense scanning showed that the target protein accounted for 19% of the total amount of the bacterial protein,and could be recognized by the T. evansi positive serum by indirect ELISA.

关 键 词：伊氏锥虫 HGPRT基因 cDNA 克隆 E.COLI 表达 核苷酸序列 PCR扩增 质粒 表达 分子量 大肠杆菌 

分 类 号：S852.729[农业科学—基础兽医学] Q78[农业科学—兽医学]