产肠毒素大肠埃希菌 K99菌毛基因的克隆与原核表达

摘要

Enterotoxigenic E.coli (ETEC)K99 complete coding frame was amplified from ETEC strain C83912 DNA with a pair of specific primers based on K99 gene.The PCR product was cloned into pMD19-T vector.Posi-tive recombinant plasmid pMD19-T-K99 was identified correctly by PCR,double-enzyme digestion and sequencing. The obtained double-enzyme digested K99 product was cloned into pET-32a(+)prokaryotic expression vector forming pET-32a(+)-K99 plasmid for expressing in BL21 (DE3).Recombinant K99 was highly expressed.West-ern-blot analysis confirmed that the recombinant K99 protein reacted specifically with K99 mono-factor seraum and immunized rabbits obtained high titer antibodies,showing its good antigenicity.The constructed ETEC K99 pro-karyotic expression plasmid and antigenicity of the recombinant K99 protein laid the foundation for K99 subunit vaccine research and K99 specific antibody preparation.%本试验采用产肠毒素大肠埃希菌（ETEC）K99菌毛基因完整编码框的特异性引物，以牛源大肠埃希菌 C83912基因组 DNA 为模板，扩增出大小为498 bp 的 ETEC K99菌毛基因完整编码框，克隆至pMD19-T 载体，阳性重组质粒 pMD19-T-K99经 PCR、双酶切鉴定和测序正确后，双酶切产物与 pET-32a （＋）载体连接，构建原核表达载体 pET-32a（＋）-K99，转化原核表达工程菌 BL21（DE3），阳性转化子经IPTG 诱导获高效表达，Western blot 证明原核表达的 K99重组蛋白能与 K99单因子血清发生特异性反应，重组蛋白免疫家兔后可产生特异性抗体，证明重组蛋白具有良好的抗原性。ETEC K99原核表达质粒的构建和表达产物抗原性研究，为进一步研究 K99亚单位疫苗以及制备 K99特异性抗体奠定了基础。