华支睾吸虫卵黄前体蛋白B基因的识别、序列分析和克隆表达

CLONING AND EXPRESSION OF A NOVEL GENE ENCODING VITELLINE PRECURSOR PROTEIN B OF CLONORCHIS SINENSIS

为识别和克隆华支睾吸虫新基因 ,对华支睾cDNA质粒文库进行随机筛选并测序 ,并利用在线生物信息学工具进行序列分析 ,识别华支睾吸虫未知基因 ,同时根据PGEX -4T- 1多克隆位点及未知基因的序列设计引物 ,PCR扩增目的基因 ,并构建原核重组质粒。结果发现了CsvpB基因 ,其完整阅读框含 76 2个碱基 ,编码 2 5 4个氨基酸 ,理论分子量为 2 7. 7kDa。序列分析表明 ,CsvpB蛋白与其它物种的卵黄前体蛋白有较高的同源性 ,所构建的重组原核表达质粒PGEX- 4T- 1 vpB经PCR、双酶切及测序证实与目标基因相符。

To identify the novel gene of Clonorchis sinensis,the cDNA library of Clornorchis sinensis was randomly screened and sequenced .The gene sequence was analyzed by tools of bioinformation online .Two primers were designed according to the cDNA sequence of the novel gene and the restriction sites of the vector of PGEX-4T-1,then the coding region of the gene was amplified by PCR and the sequence was cloned in the vector. The recombinant of PGEX-4T-1-Cs vpB has been identified by PCR and by restricted enzymes. As result,the full-length cDNA of Cs vpB found from the cDNA library includs 762bps encoding 254 amino acids,and molecule weight of the putative protein is 27.7kDa.Sequence analysis showed that the protein of Cs vpB has a high homology with that of other species.