华支睾吸虫表膜相关蛋白基因的扩增、序列分析和克隆

Amplification,sequence analysis and cloning of a novel gene encoding tegumental protein of Clonorchis sinensis

目的识别华支睾吸虫新基因,并将所发现的华支睾吸虫表膜相关蛋白(tegumental protein ofClonorchis sinensis,CsTP)的cDNA序列克隆到原核载体中,为进一步的研究奠定基础。方法对华支睾cDNA质粒文库进行随机筛选并测序,用在线生物信息学工具进行序列分析,识别CsTP基因,并对其进行同源性、物理性质及结构分析。设计引物,从华支睾cD-NA质粒文库中扩增目的基因cDNA序列,并构建原核重组质粒,相同引物从华支睾吸虫基因组中扩增出CsTP的DNA序列。结果发现CsTP基因,其cDNA完整阅读框含555个碱基,编码184个氨基酸,理论分子量为20.7kDa;其DNA序列含1393个碱基,含有3个外显子、2个内含子。序列分析表明,CsTP蛋白与其它物种的表皮蛋白或膜相关蛋白有一定的同源性。所构建的重组原核表达质粒PGEX-4T-1-CsTP经PCR、双酶切及测序证实与目的基因相符。结论发现华支睾吸虫表膜相关蛋白基因,成功构建其重组原核表达质粒为进一步研究该基因的功能提供了条件。

To identify the novel gene of Clonorchis sinensis, and to clone the gene encoding tegumental protein （CsTP） into the prokaryotie expession vector PGEX-4T-1, the cDNA library of Clornorchis siensis was randomly screened and sequenced. The gene sequence was analyzed by tools of bioinformation online. Two primers were designed according to the 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-CsTP had been identified by PCR and by restricted enzymes.The DNA sequence of CsTP was amplified by PCR from genome of Cs. The full-length cDNA of CsTP was found from the cDNA library including 555bps encoding 184 amino acids, and molecular weight of the putative protein was 20.7KDa.DNA sequence of CsTP included 1393bps,3 extrons and 2 introns.Sequenee analysis showed that the protein of CsTP had a high homology with that of other species. A novel gene coding CsTP was found and PGEX-4T-1-CsTP was constructed successfully.