真核重组质粒pCDNA3.1-Flag-PTEN 和原核重组质粒pGEX-6P-1-GST-PTEN构建  

Construction of eukaryotic recombinant plasmid pCDNA3.1-Flag-PTEN and prokaryotic recombinant plasmid pGEX-6P-1-GST-PTEN

作　　者：柴云 于明[1] 朱颖[1] CHAI Yun;YU Ming;ZHU Ying(Department of Neurology,The Affiliated Hospital of Jiangsu University,Zhenjiang Jiangsu 212001,China)

机构地区：[1]江苏大学附属医院神经内科

出　　处：《蚌埠医学院学报》2019年第11期1437-1440,共4页Journal of Bengbu Medical College

基　　金：国家自然科学基金资助项目(81601043);镇江市重点研发计划项目(SH2018037)

摘　　要：目的:构建人第10号染色体缺失的磷酸酶(PTEN)真核重组质粒pCDNA3.1-Flag-PTEN和原核重组质粒pGEX-6P-1-GST-PTEN,研究PTEN融合质粒生物学效应。方法:根据基因库中PTEN序列,设计并合成相关引物。用基因重组技术将目的片段插入pCDNA3.1-Flag和pGEX-6P-1-GST空载质粒中,通过PCR和DNA测序,鉴定插入基因序列。将pCDNA3.1-Flag-PTEN转染到HEK 293细胞,将pGEX-6P-1-GST-PTEN转到E.coli BL-21中,通过Western blotting和考马斯亮蓝染色法分别检测PTEN蛋白的表达。结果:PCR显示目的基因插入成功;DNA测序显示插入序列与目的基因序列完全一致,无位点突变;Western blotting和考马斯亮蓝染色法证实融合质粒可以在生物体内正确表达。结论:成功构建PTEN相关真核和原核融合质粒并正确表达,为PTEN在帕金森疾病中的机制研究奠定了基础。Objective:To construct the eukaryotic recombinant plasmid pCDNA3.1-Flag-PTEN and prokaryotic recombinant plasmid pGEX-6 P-1-GST-PTEN of phosphatase and tension homologue deleted on chromosome ten(PTEN),and study the biological effects of PTEN fusion plasmid.Methods:The primers of PTEN gene were designed according to the sequence in Gene Bank.The target fragment was inserted into pCDNA3.1-Flag and pGEX-6 P-1-GST empty vector by genetic recombination technique,and the inserted gene sequence was identified by PCR and DNA sequencing.The pCDNA3.1-Flag-PTEN was transfected into HEK293 cells and pGEX-6 P-1-GST-PTEN was transfected into Escherichia coli BL-21.The expression of PTEN protein was detected by Western blot and Coomassie brilliant blue staining.Results:The PCR results showed that the target gene was successfully inserted.The results of DNA sequencing showed that the inserted sequence was completely consistent with the target gene sequence,and no site-mutation was found.The results of Western blot and Coomassie brilliant blue staining confirmed that the fusion plasmid could be correctly expressed in vivo.Conclusions:The successful construction and correct expression of PTEN in eukaryotic and prokaryotic expression vectors can provide a basis for further study of the mechanism of PTEN in Parkinson′s disease.

关 键 词：抑癌蛋白 人第10号染色体缺失的磷酸酶 真核 原核 克隆构建 重组表达 

分 类 号：R392.13[医药卫生—免疫学]