华支睾吸虫表膜相关抗原重组表达载体的构建

目的通过筛选cDNA文库识别华支睾吸虫新基因,并构建20.8?kDa华支睾吸虫表膜相关抗原(CSTA20.8)重组表达载体,为进一步研究其功能及其应用奠定基础.方法对华支睾吸虫cDNA文库进行筛选,通过NCBI和ExPasy网站的Blast程序进行序列比对,识别华支睾吸虫新基因,并应用Motifscan、NCBI Conserved Domain Search等程序对其进行结构域分析.将所发现的华支睾吸虫表膜相关抗原基因编码区定向克隆到原核及真核表达载体PGEX-4T-1和PcDNA3上,构建的PGEX-4T-1-CSTA20.8、PcDNA3-CSTA20.8重组表达质粒经PCR、双酶切及测序证实.结果发现华支睾吸虫表膜相关抗原基因,完整阅读框含555个碱基,编码184个氨基酸,理论分子量为20.8kDa,理论pI为4.33.序列分析表明,华支睾吸虫CSTA20.8编码氨基酸序列与其它物种有较高的同源性,CSTA20.8具有完整的钙结合蛋白保守功能域.所构建的重组原核和真核表达质粒经PCR、双酶切及测序证实与目标基因相符.结论发现华支睾吸虫表膜相关抗原基因,并成功构建原核和真核重组表达质粒.

Construction and analysis on recombination expression vector of tegument membrane-associated antigen from clonorchiasis sinensis

Objective To identify the novel genes of clonorchiasis sinensis by screening the cDNA library and clone the screened gene-20.8kDa te gume nt membrane-associated antigen(CSTA20.8) to the prokaryotic expression vectors and eukarytoyic expression vectors.Methods The cDNA library of clonorchiasis sinensis we re s creened.The homologue of the novel sequences with a high identity was compared o n amino acid and nucleotide level with blast programme on NCBI BLAST site.The mo tifs of the protein coded by the novel gene were searched with MotifScan in a pr oteins sequence on ExPASy site and NCBI Conserved Domain Search.Bisides,a pair o f specific oligonucleotide primers via Ecoll,Xholl restriction sites respect ly w ere designed and synthysed according to the coding region of CSTA208 gene found by screening library.The coding region of CSTA20.8 gene was amplified by PCR an d then cloned into the prokaryotic expression vectors PGEX-4T-1 and eukaryotyic expression vectors PCDNA3 via Ecoll and Xholl restriction sites,and transfor med into E.coli BL21 respectively.The positive recombinant PGEX-4T-1-CSTA20.8-and P CDNA3-CSTA20.8 were screened and identified by endonuclease digestion,PCR and s equence.Results A novel cDNA sequence coding CSTA20.8 was found from the cDNA library of clonorchiasis sinensis.Translation of nucleotides s eque nce revealed putative open reading frame of 184 amino acids with a molecular mas s of 20.767?5?Kd and PI of 4.33.The predicted primary structure of CSTA 20. 8 shared high sequence homology with other species and contained conserved doma in of Efh Motif.The recombinant plasmids PGEX-4T-1-CSTA20.8 and PCDNA3-CSTA20.8 w ere constructed.Conclusion A novel gene coding CSTA20.8 of clonorchias is sinensis was found and cloned and its prokaryotic expression vectors and eu ka ryotyic expression vectors were constructed successfully.