Flag-ALD融合载体构建及蛋白表达  

Reconstruction and Protein Expression of Flag-ALD Fusion Vector

作　　者：曹欣 唐燕琼[1] 李宏[1] 马香[1] 刘柱[1] CAO Xin;TANG Yanqiong;LI Hong;MA Xiang;LIU Zhu(Key Laboratory of Tropical Biological Resources of Ministry of Education/School of Life and Pharmaceutical Sciences,Hainan University,Haikou,Hainan 570228,China)

机构地区：[1]海南大学生命科学与药学院/热带生物资源教育部重点实验室,海口570228

出　　处：《热带生物学报》2020年第2期132-137,155,共7页Journal of Tropical Biology

基　　金：海南省自然科学基金青年基金(319QN161);海南省自然科学基金(317015);国家自然科学基金项目(31560021,31772887,31860676,31960027)。

摘　　要：维氏气单胞菌(Aeromonas veronii)是感染鱼类的最常见致病菌之一,为了解维氏气单胞菌中丙氨酸脱氢酶(alanine dehydrogenase,ALD,EC 1.4.1.1)的调控功能,笔者通过分子克隆获得Flag-ALD融合表达载体,并在大肠杆菌中大量表达该蛋白。即以维氏气单胞菌C4基因组DNA为模板,采用PCR方法扩增获得N端带有Flag标签的ald基因,将带有标签的目的基因与原核表达载体pACYCDuet连接,并将重组质粒pACYCDuet-Flag-ald转化到大肠杆菌(E.coli)BL21中。添加异丙基-β-D-1-硫代半乳糖吡喃糖苷(Isopropylbeta-D-thiogalactopyranoside,IPTG)诱导重组蛋白大量表达。SDS-PAGE分离与Western blot结果表明,来源于维氏气单胞菌的Flag-ALD重组蛋白在E.coli中以可溶性形式成功表达。本实验构建的Flag标记靶标蛋白可为研究丙氨酸脱氢酶在维氏气单胞菌中的功能以及在生物抑菌剂方面的应用提供技术支持。Alanine dehydrogenase(ALD,EC 1.4.1.1)is a microbial enzyme,which plays an important role in the transformation of alanine to pyruvate,carbon metabolism,nitrogen metabolism,and energy metabolism in microorganism.In order to study the function of ALD protein in Aeromonas veronii that causes important aquatic diseases,C4 genomic DNA of A.veronii was used as a template to amplify ALD gene with Flag tag on the N-terminal by PCR.The target gene was incorporated into the expression vector pACYCDuet,and the recombinant plasmid pACYCDuet-Flag-ALD was transformed into E.coli BL21.The recombinant protein was induced by Isopropyl-beta-D-thiogalactopyranoside(IPTG).SDS-PAGE and Western blot showed that the recombinant protein of Flag-ALD was successfully expressed in a soluble form in E.coli.The results laid a foundation for the expression of exogenous ALD protein.The flag labeled target protein may provide a technical support for the further study on the function of alanine dehydrogenase in A.veronii and the development of bacteriostatic agents.

关 键 词：丙氨酸脱氢酶 基因克隆 重组质粒 蛋白表达 

分 类 号：Q782[生物学—分子生物学]