亚洲牛带绦虫TaCRISP基因克隆、表达和序列分析

目的通过筛选亚洲牛带绦虫(Taenia saginata asiatica)成虫cDNA质粒文库,识别半胱氨酸分泌蛋白(Ta CRISP),并进行克隆表达,为进一步研究其功能提供基础依据。方法用生物信息学方法从亚洲牛带绦虫成虫全长cDNA质粒文库中识别TaCRISP的全长编码基因并预测编码蛋白质的各种结构与功能;将其编码区序列克隆到原核表达载体pET-30a(+)上,测序鉴定重组质粒,之后进行诱导表达,表达产物经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定。结果该基因全长1090bp,编码239个氨基酸,1aa-16aa位为分泌信号肽,理论分子量为26973.8,等电点为5.96。生物信息分析揭示,Ta CRISP与中殖孔绦虫CRISP一致性为38%,相似性为54%;所构建的重组体经PCR、双酶切及测序鉴定与目标基因相符。SDS-PAGE结果表明,目的基因在大肠埃希菌BL-21/DE3中表达成功。结论发现亚洲牛带绦虫Ta CRISP基因,成功构建重组原核表达质粒并表达出融合蛋白。

Cloning,expression and sequence analysis of cysteine-rich secretion protein 2 gene from Taenia saginata asiatica

ObjectiveTo search and identify novel gene by screen ing the cDNA library of adult Taenia saginata asiatica, and to clone and express the gene so as to lay a foundation for the further study of Ta CRISP gene's function.MethodsBy the bioinformatics analyzing tools in bioinformatics webs site,a Ta CRISP 2 full-length gene from the Ta eniasa ginata asiatica full-length cDNA plasmid libratory was identified and the coding region sequence and the characteristics of the deduced prote in were analyzed.The coding region of Ta CRISP was amplified with PCR,endonuc lease digestion and cloned in to the prokaryotic expression vector pET-30a(+)and then expressed in E.coli BL 21 with IPTG induction.The recombinant protein was detected by SDS-PAGE.ResultsA novel cDNA sequence encoding Ta CRISP with a molecular mass of 269738,pI 5.96,which was 1090 bp and encoding 239 aa,and with 1aa to 16aa signal peptide,was found from the cDNA library of Taenia saginata asiatica.The recom binant plasmid pET-30a(+)-TaCRISP was constructed and expressed in fusion prote in.ConclusionA novel gene encoding TaCRISP of Taenia saginata asiatica was identified and its prokaryotic expression vectors was expressed in fusion protein successfully.