大肠杆菌O157∶H7独有基因z3276的表达及初步鉴定  

Preliminary identification and expression of unique gene z3276 of Escherichia coli O157∶H7

作　　者：张碧成[1,2] 张雪寒[2] 何孔旺[2] 范红结[1] 

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室/国家兽用生物制品工程技术研究中心,江苏南京210014

出　　处：《中国兽医学报》2016年第8期1354-1357,共4页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31201919);农业部"948"计划资助项目(2014-S17);江苏省农业科技自主创新资金资助项目(CX(13)5033)

摘　　要：经BLAST分析表明,开放阅读框z3276是大肠杆菌O157∶H7独有的遗传标志性基因,编码氨基酸与大肠杆菌Ⅰ型菌毛有较高的同源性,但z3276基因编码蛋白的生物学特性尚不明确。为了明确z3276基因编码蛋白的抗原性,进一步研究其在大肠杆菌O157∶H7致病过程中的作用。采用PCR方法扩增z3276基因,去除其信号肽序列,克隆到原核表达载体pColdⅠ,转化感受态细胞BL21获得阳性重组菌,IPTG诱导表达重组蛋白,以大肠杆菌O157∶H7全菌抗血清为一抗,Western blot方法检测重组z3276蛋白抗原性。结果显示,成功构建重组菌BL21(pColdⅠ-z3276),并获得高效表达;相对分子质量33 000,占菌体总蛋白30%以上,主要以包涵体形式存在于菌体沉淀中;且与兔源全菌多克隆抗血清发生特异性反应,条带明显。The BLAST analysis showed that a unique open reading frame （ORF）,z3276 ,is a genetic marker gene in Escherichia coli （E. coli ） O157 ： HT. The protein encoding by the z3276 gene （named Z3276 protein） has high homology with Escherichia coli type I pili, but its biological characteristics is unknown. To test the antigenicity of the z3276 protein which also lays the foun- dation for the further study on its roles in E. coli O157 ： H7 pathogenesis. The z3276 gene frag- ment without the signal peptide sequence was amplified by conventional PCR, cloned into the pro karyotic expression vector pCold I ,and transformed into complement cells of E. coli BL21 to con- struct the recombinant positive strain. The recombinant protein expression was induced under IPTG and identified antigenicity by Western blot assay using anti E. coli O157 ：H7 sera from rab- bits. The results showed that recombinant strain BL21 （pCold I-z3276） was constructed success- fully,the z3276 protein of 33 000 was expressed efficiently,accounting about 30% of total bacterial protein,as a form of inclusion bodies in the bacterial pellets. Western blot assay showed that the recombinant z3276 protein could react to rabbit polyclonal antiserum against E. coli O157 ： H7 with distinct specific bands.

关 键 词：大肠杆菌O157:H7 标签基因 z3276基因 原核表达 

分 类 号：S855.1[农业科学—临床兽医学] R535[农业科学—兽医学]