牛Ⅱ型链球菌van B2蛋白的原核表达及纯化

Prokaryotic expression and purification of van B2 protein of Streptococcus bovis biotypeⅡ

目的原核表达并纯化牛Ⅱ型链球菌(Streptococcus bovis biotypeⅡ)van B2蛋白。方法采用PCR法从牛Ⅱ型链球菌基因组DNA中扩增van B2基因,克隆至原核表达载体pET-28a(+)中,构建重组表达质粒pET-28a-van B2,转化大肠杆菌Rossata(DE3),IPTG诱导表达。表达的重组蛋白经Ni柱亲和层析纯化后,进行SDS-PAGE及Westernblot分析。结果 PCR扩增获得597 bp的van B2基因片段;重组表达质粒pET-28a-van B2经双酶切及测序证明构建正确;表达的重组van B2蛋白相对分子质量约为29 000,主要以可溶性形式表达;纯化的重组蛋白纯度为70%,可被小鼠抗牛链球菌血清Ⅱ型多克隆抗体特异性识别。结论原核表达并纯化了牛Ⅱ型链球菌van B2蛋白,为van B2基因的耐药性研究奠定了物质基础。

Objective To express the van B2 protein of Streptococcus bovis biotypeⅡ and purify the expressed product.Methods The van B2 gene was amplified from genomic DNA of S.bovis biotype Ⅱ by PCR,and cloned into prokaryotic expression vector pET-28a（＋）.The constructed recombinant plasmid pET-28a-van B2 was transformed to E.coli Rossata（DE3） for expression under induction of IPTG.The expressed recombinant protein was purified by nickel ion affinity chromatography,and identified by SDS-PAGE and Western blot.Results The van B2 gene fragment at a length of 597 bp was amplified by PCR.Restriction analysis and sequencing proved that recombinant plasmid pET-28a-van B2 was constructed correctly.The expressed recombinant van B2 protein,with a relative molecular mass of about 29 000,mainly existed in a soluble form and reached a purity of 70% after purification,which was recognized specifically by polyclonal antibody against S.bovis biotypeⅡ.Conclusion The van B2 protein of S.bovis was expressed in prokaryotic cells and purified,which laid a foundation of study on drug-resistance of van B2 gene.