重组胸腺素α1 pMAL-C2x-Tα1/TB1工程菌的构建与表达  

Construction and Expression of Recombinant Tα1 pMAL-C2x-Tα1/TB1 Engineering Strain

作　　者：刘中禄[1] 陶翠兰[1] 莘旭妮[2] 李树民[2] 

机构地区：[1]第三军医大学大坪医院野战外科研究所实验动物中心,重庆400042 [2]解放军军事医学科学院军事兽医研究所,长春130062

出　　处：《中国比较医学杂志》2012年第7期13-16,共4页Chinese Journal of Comparative Medicine

基　　金：重庆市科技攻关计划项目(30111-14281)

摘　　要：目的构建表达重组胸腺素α1(Tα1)的pMAL-C2x-Tα1/TB1工程菌。方法将人工合成的Tα1序列进行PCR扩增,将扩增的片段和pMAL-C2x质粒载体分别经BamHI和EcoR I双酶切后,用T4 DNA快速连接酶连接构建pMAL-C2x-Tα1融合表达质粒,再经测序正确后,将重组体转化至大肠埃希菌TB1菌中,pMAL-C2x-Tα1/TB1菌在LB液体培养基中培养,经IPTG诱导表达麦芽糖结合蛋白与Tα1的融合蛋白(MBP-Tα1),采用Westernblot对MBP-Tα1进行鉴定。结果 pMAL-C2x-Tα1/TB1工程菌能有效表达MBP-Tα1,融合蛋白占菌体蛋白的33.6%,分子量约为45×103。结论工程菌的成功构建和表达为重组Tα1的纯化、生物学活性等研究奠定了基础。Objective To construct an engineering strain of pMAL-C2x-Tα1/TB1 which can express Thymosin α1（Tα1）.Methods Tα1 gene was synthesized and amplified by PCR,PCR product and pMAL-C2x vector were digested with restriction endonuclease BamHI and EcoR I respectively,and were linked with T4 DNA ligase to construct pMAL-C2x-Tα1.After being sequenced,pMAL-C2x-Tα1 vector was transformed into E.coli TB1 for fusion expression under induction of IPTG..The expressed product was identified by Western blot.Results The DNA sequence of the synthesized Tα1 gene was identical to the original design.The constructed recombinant plasmid pMAL-C2x-Tα1 was highly expressed in E.coli TB1.The BMP-Tα1 fusion protein was about 33.6% of the total bacteria protein and the relative molecular weight of BMP-Tα1 fusion protein was about 45×103.Conclusion The successful construction and expression of pMAL-C2x-Tα1/TB1 laid a foundation for the research of purification and biological acitivity of recombinant Tα1.

关 键 词：重组胸腺素α1 基因克隆 质粒pMAL-C2x 融合表达 

分 类 号：S859[农业科学—临床兽医学] R332[农业科学—兽医学]