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志贺样毒素IIeA酶活性位点基因突变株的构建与表达

Mutation and Expression of A Subnit of Shiga-Like Toxin Type II Variant

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【作者】 徐建生成大荣陈雷董国雄李俊宝

【Author】 XU Jian-sheng, CHENG Da-rong, CHEN Lei, DONG Guo-xiong LI Jun-bao (Vet Med Coll, Yangzhou Univ, Yangzhou, 225009, China)

【机构】 扬州大学兽医学院扬州大学兽医学院 江苏扬州225009江苏扬州225009

【摘要】 利用重叠延伸PCR法对志贺样毒素 型变异体A亚单位(SLT- eA)基因进行修饰扩增,将第167位编码Glu的密码子突变为Gln的密码子,第170位编码Arg的密码子突变为编码Lys密码子,并用质粒载体pGEX-6P-1在大肠杆菌BL21中进行融合表达,在不改变其蛋白空间构象的同时降低其毒性。重组SLT- eA突变株pGEX-Amu的大量表达为研究SLT- eA在体内的生物学特性提供依据。

【Abstract】 Shiga-like toxin typeⅡvariant (SLT-Ⅱe) is composed of one A subunit and five B subunits. The A subunit possesses RNA N-glycosidase activity, which specifically removes an adenine in the 28S subunit of eukaryotic rRNA, leading to inactivation of the cellular protein synthesis and disorganization of metabolic functions of the cell. The double points mutated at amino acid residues 167(Glu-Gln) and 170(Arg-Lys) of SLT-IIeA by the technology of SOE (Splicing by overlap extension). The product was cloned into prokaryotic expression vector pGEX-6P-1 and resulting recombinant was called pGEX-Amu. The recombinant plasmid was transformed into E.coli BL21 and GST-fusion protein (GST-ⅡeA) with respected size was expressed by Western-blotting using monoclonal antibody specific for SLT-ⅡeA.

【基金】 江苏省自然科学基金资助项目(BK95087303)
  • 【文献出处】 扬州大学学报 ,Journal of Yandzhou University , 编辑部邮箱 ,2004年04期
  • 【分类号】S852.61
  • 【被引频次】1
  • 【下载频次】77
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