南方医科大学学报

南方医科大学学报 ›› 2013, Vol. 33 ›› Issue (12): 1757-.

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小鼠生精相关基因pQE/Dnajb13重组载体的构建和蛋白表达

朱莉，刘刚   

  • 出版日期:2013-12-20 发布日期:2013-12-20

Construction of pQE/Dnajb13 recombinant plasmid and the protein expression

  • Online:2013-12-20 Published:2013-12-20

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摘要： 目的利用分子克隆技术构建重组表达载体pQE/Dnajb13，在大肠埃希菌中高效表达、鉴定并纯化。方法应用RT-PCR技
术，以小鼠睾丸cDNA文库为模板扩增Dnajb13基因全长开放阅读框，将其连接到pQE载体后测序鉴定；将重组质粒转入宿主菌
E.coli M15，经IPTG诱导表达His/Dnajb13融合蛋白，Ni2+亲和层析纯化融合蛋白，采用SDS-PAGE 和Western blotting 对融合蛋
白进行分析和鉴定。结果成功构建了pQE/Dnajb13重组质粒，测序结果与预期一致；转化重组质粒的E.coli M15经IPTG 37 ℃
诱导4 h后高效表达His/Dnajb13融合蛋白。结论成功进行了pQE/Dnajb13重组质粒的构建和重组蛋白的表达，为Dnajb13基因
的功能研究奠定了基础。

Abstract: Objective To construct pQE/Dnajb13 recombinant vector and induce the expression of the fusion protein. Methods
The open reading frame (ORF) of Dnajb13 gene was amplified from mouse testis cDNA library by PCR. The products were
digested by SacI and SalI and subcloned into pQE vector. After identification by DNA sequence analysis, the recombinant
plasmids were transformed into competent E.coli M15 cells. The His/Dnajb13 fusion protein was expressed with IPTG
induction and purified with Ni2+ affinity chromatography. Western blotting was used to detect Dnajb13 expression. Results The
recombinant vector pQE/Dnajb13 was successfully constructed. His/Dnajb13 fusion protein was expressed abundantly at 4 h
after IPTG induction, and Western blot analysis demonstrated the presence of Dnajb13 expression in E.coli M15. Conclusion
We have successfully constructed pQE/Dnajb13 recombinant vector, which may facilitate further investigation of the role of
Dnajb13 in spermatogenesis.