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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 and purify van B2 protein of Streptococcus bovis biotype Ⅱ. Methods The van B2 gene was amplified by PCR from genomic DNA of Streptococcus bovani Ⅱ and cloned into prokaryotic expression vector pET-28a (+). The recombinant plasmid pET-28a-van B2 was transformed into E. coli Rossata (DE3) , IPTG induced expression. The expressed recombinant protein was purified by Ni column affinity chromatography and analyzed by SDS-PAGE and Western blot. Results A 597 bp fragment of van B2 gene was obtained by PCR. The recombinant plasmid pET-28a-van B2 was proved to be correct by double enzyme digestion and sequencing. The relative molecular mass of recombinant van-B2 protein was approximately 29 000, mainly in soluble form The purity of purified recombinant protein was 70%, which could be specifically recognized by mouse anti-Streptococcus pneumoniae serotype II polyclonal antibody. Conclusions The prokaryotic expression and purification of van B2 protein of van B2 can lay a material foundation for the study of drug resistance of van B2 gene.