亚洲牛带绦虫自吞噬相关蛋白3基因克隆及表达

Cloning,expression and sequence analysis of autophagy-related protein 3(APG3-like)gene from Taenia asginata asiatica

目的从亚洲带绦虫成虫cDNA质粒文库中识别并预测自吞噬相关蛋白（autophagy related protein3-like,APG3）基因,并将该基因进行克隆表达,为进一步研究其生物学功能提供基础依据。方法利用生物信息网站及其他分析软件包,分析该蛋白质理化特性等,并将其克隆到原核表达质粒pET-28a（＋）中,经PCR、双酶切及测序鉴定之后诱导表达,表达产物通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行鉴定。结果该基因全长为1 331 bp,编码区为92-1099,编码335个氨基酸,理论分子量、等电点分别为37 498.5 Da和4.71。PCR、双酶切及DNA测序结果均表明pET-28a（＋）-Ta APG3重组质粒构建成功。经过尿素破包涵体法得到纯化蛋白。结论发现亚洲牛带绦虫APG3基因,成功构建重组原核表达质粒并表达出融合蛋白。

Objective To search and identify novel gene by screening the cDNA library of adult Taenia saginata asiatica and to insert the screened gene into autophagy-related protein 3（APG3）,then to analyze and predict the structure and characteristics of Ta.APG3 gene by bioinformatics.Methods By the bioinformatics analyzing tools and combining other complicated bioinformatics software package,the characteristics of the protein was analyzed.The gene was inserted into the prokaryotic expression vectors pET-28a（＋）,then was amplified with PCR and endonuclease digestion and then expressed with IPTG induction.The recombinant protein was detected by SDS-PAGE.Results The novel cDNA sequence encoding Ta.APG3 has a molecular mass of 37498.5,and an isoelectric point of 4.71.The gene is 1331bp coding 332 amino acids.PCR,double enzyme digestion and DNA sequencing all indicated that pET-28a（＋） and APG3 recombinant plasmid was successfully constructed.SDS-PAGE results showed that the gene expression took place in Escherichia coli BL21/DE3,and highly pure protein was achieved after urea inclusion body deposits.Conclusion A novel gene of Taenia saginata asiatica was constructed in prokaryotic expression vector and its fusion protein was expressed successfully.