犬弓首蛔虫卵黄原蛋白DUF1943结构域的克隆及原核表达  

Cloning and Prokaryotic Expression of the DUF1943 Domain of Toxocara canis Vitellogenin

作　　者：罗永莉 朱宏宏 江艾耘 罗永芳[1] 旷策嫣 周荣琼[1] 

机构地区：[1]西南大学动物科学学院,重庆荣昌402460

出　　处：《西南大学学报（自然科学版）》2018年第1期15-20,共6页Journal of Southwest University(Natural Science Edition)

基　　金：国家自然科学基金项目(31172313);中央高校基本科研业务费专项资金项目(XDJK2016E087)

摘　　要：为克隆犬弓首蛔虫Toxocara canis,T.canis卵黄原蛋白DUF1943结构域(The domain of unknown function1943,DUF1943)(Tc-duf1943),构建其原核表达载体pET-28a(+)/duf1943,并检测重组蛋白在大肠杆菌Escherichia coli,E.coli中的表达形式.该文根据T.canis的转录组数据,以T.canis雌、雄虫cDNA为模板扩增Tcduf1943,然后与表达载体pET-28a(+)连接,转化E.coli BL21(DE3),经IPTG诱导,对目的蛋白的原核表达形式和蛋白质纯化进行鉴定.结果显示Tc-duf1943序列长度为864bp,含有一个完整的开放阅读框,编码288个氨基酸;SDS-PAGE电泳结果表明,重组蛋白以包涵体形式存在,相对分子质量大小约为3.5×10~4;对表达条件进行优化后,以0.4mmol/L IPTG,于37℃诱导4h时可获得大量目的蛋白;利用Ni-NTA亲和层析柱纯化可以获得具有较高纯度的目的蛋白.该试验成功构建了pET-28a(+)/duf1943原核表达载体,为进一步研究TcDUF1943的生物学功能奠定了基础.To clone the DUF1943（the domain of unknown function 1943）of vitellogenin in Toxocara canis,the prokaryotic expression vector pET-28 a（＋）/duf1943 of Tc-duf1943 was constructed and its expression form in Escherichia coli was detected.According to the transcriptome data about T.canis,and using the cDNA of female and male as templates,Tc-duf1943 was cloned successfully.Then the target gene was connected with the prokaryotic expression vector pET-28 a（＋）（pET-28 a（＋）/duf1943）,E.coli BL21（DE3）was transformed and the prokaryotic expression and purification of the protein was identified.The results showed that Tc-duf1943 was 864 bp in length,with a complete open reading frame,encoding 288 amino acids,and SDS-PAGE indicated that the recombinant protein was expressed in inclusion bodies,with a molecular weight of about 3.5×10~4,The optimal condition of prokaryotic expression was induced by 0.4 mmol/L IPTG for 4 hours at 37℃ .Purified with Ni-NTA affinity chromatography,target protein with high purity was obtained.In conclusion,this experiment successfully constructed the prokaryotic expression vector of pET-28 a（＋）/duf1943,which laid a foundation for further biological function study of TcDUF1943.

关 键 词：犬弓首蛔虫 DUF1943 克隆 原核表达 

分 类 号：S858.292[农业科学—临床兽医学]