胸腺素α1重组原核表达质粒的克隆及诱导表达  

Cloning and expression of the thymosin α1 recombinant procaryotic plasmid

作　　者：彭仕芳[1] 傅蕾[1] 谭德明[1] 苏先狮[2] 

机构地区：[1]中南大学湘雅医院感染病科,湖南长沙410008 [2]中南大学湘雅二医院感染病科

出　　处：《中国医师杂志》2007年第9期1190-1192,共3页Journal of Chinese Physician

摘　　要：目的：构建胸腺素α1（Tα1）基因的原核表达质粒，并诱导表达。方法：根据Tα1的DNA序列设计引物，采用PCR法扩增Tα1的cDNA，将其克隆入T-vector，形成中介重组体pT—Tα1，再将其亚克隆至原核表达载体pGEX-3X，构建重组质粒pGEX-3X-Tα1，用内切酶酶切鉴定及DNA测序分析；将pGEX-3X-Tα1转入大肠杆菌，用RT-PCR方法检测宿主菌中Tα1mRNA，经IPTG诱导，用SDS—PAGE检测有无相应大小的Tα1融合蛋白表达。结果：经DNA测序和SDS—PAGE法鉴定，成功构建了Tα1重组质粒基因工程菌，表达和纯化了具有Tα1融合蛋白。结论：构建的Tα1重组质粒基因工程菌，表达和纯化了具有Tα1融合蛋白，为今后的研究打下了基础。Objective To construct the recombinant prokaryotic expression plasmid of TotI and induce its expression in E. Coli, Methods According to the DNA sequence of Tα1, a pair of primers were designed and synthesized, Tα1 cDNA was amplified by polymerase chain reaction （PCR）. Then the PCR product was cloned in T-vector to construct intermediate recombinant pT-Tα1 and sub-cloned in prokaryotic expression plasmid to construct pGEX-3X-Tα1, The cloning result was analyzed by double restriction endonuclease reaction and DNA sequencing. After transformation and expression induction in E, Coli, RT-PCR and SDS-PAGE were performed to detect Tα1 mRNA and goal fusion protein, TotI cDNA was obtained by PCR, Results Double restriction endonuclease reaction and DNA sequencing results showed that the cloning of pGEX-3X-Tα1 was successful, SDS-PAGE results showed that the expected Tα1 fusion protein was expressed, Conclusion We obtained the recombinant plasmid pGEX-3X-Tα1. The TotI fusion protein was expressed in E. Coli JM109 and confirmed by SDS-PAGE, which established basic for further study.

关 键 词：胸腺素/遗传学 质粒 

分 类 号：Q[生物学]