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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 the open reading frame z3276 is a unique genetic marker gene of E. coli O157: H7. The encoded amino acids have high homology with E. coli type Ⅰ pili, but the biological characteristics of z3276 gene unclear. In order to clarify the antigenicity of z3276 gene encoding protein, further study its role in the pathogenic process of E. coli O157: H7. The z3276 gene was amplified by PCR and its signal peptide sequence was removed. The recombinant plasmid was cloned into prokaryotic expression vector pColdⅠ. The competent cells were transformed into BL21 to obtain recombinant plasmids. The recombinant protein was induced by IPTG. The E. coli O157: H7 The antigenicity of recombinant z3276 protein was detected by Western blot. The results showed that the recombinant bacterium BL21 (pColdⅠ-z3276) was successfully constructed and expressed efficiently. The relative molecular mass was 33 000, accounted for more than 30% of the total bacterial protein, mainly in the form of inclusion bodies in the bacterial pellet. Polyclonal whole-cell polyclonal antiserum specific reaction, obvious band.