抗菌肽PR-39真核表达载体的构建及表达检测

Construction of eukaryotic expression vector encoding antimicrobial peptides PR-39 and its expression in 293T cell

目的:构建富含脯氨酸精氨酸的39位氨基酸抗菌肽(Proline-arginine rich 39-amino acid peptide,PR-39)的真核表达载体,并检测它在293T细胞中的表达情况。方法:以含有PR-39核心编码区(Core coding regions,CDS)序列的质粒pBluescriptⅡSK-PR-39为模板,PCR扩增PR-39CDS序列并测序。扩增片段插入真核表达质粒pGC-FU中,构建包含PR-39基因的重组表达质粒pGC-FU-PR-39。阳性克隆用限制性内切酶酶切及测序鉴定后用脂质体2000转染293T细胞,荧光显像观察其转染效率,Western blot检测目的蛋白表达情况。结果:成功扩增出PR-39基因,通过酶切测序鉴定证明成功构建了包含PR-39的真核表达载体pGC-FU-PR-39,转化293T细胞24h后观察到荧光,Western blot检测到目的蛋白。结论:本实验成功构建PR-39真核表达载体,并能在293T细胞中表达PR-39-GFP融合蛋白,这为进一步研究PR-39的作用奠定了基础。

Objective： To construct eukaryotic plasmid expressing PR-39 and test whether the plasmid can be expressed in the 293 T cell. Methods：The core coding regions （CDS） sequence of PR-39 was amplified by PCR from pBluescript II SK-PR-39.Then the PR-39 gene was inserted into the eukaryotic expression plasmid pGC-FU. The resultant recombinant plasmid was confirmed by restriction enzyme cutting and sequencing, and then was designated as pGC-FU-PR-39. The recombinant plasmid pGC-FU-PR-39 was transfected into 293 T cell by Lipofectamine 2000. Transfected efficiency was observed by fluorographic unit, Western blot was used to certify whether the purpose protein was correctly expressed in 293 T cell. Results：The CDS sequence of PR-39 was correctly amplified. The recombinant plasmid was successfully constructed and could be correctly expressed in 293 T cell. Conclusion：We successfully constructed PR-39 eukaryotic expression plasmid and observed the expression of PR-39-GFP fusion protein. It lays a foundation for further studies of PR-39.