牛种布鲁菌VirB12蛋白的原核表达及纯化

目的原核表达并纯化牛种布鲁菌(Brucella)VirB12蛋白。方法利用PCR法从牛种布鲁菌基因组中扩增VirB12基因,插入pET-30a(+)载体,构建重组表达质粒pETV12,转化大肠埃希菌BL21(DE3),IPTG诱导表达。表达的重组蛋白经组氨酸结合树脂柱纯化后,进行SDS-PAGE及Western blot分析。结果重组表达质粒pETV12经双酶切及测序证明构建正确;表达的重组蛋白相对分子质量约22 000,纯化的重组蛋白纯度达94%,可被布鲁菌免疫兔血清特异性识别。结论原核表达并纯化了牛种布鲁菌VirB12蛋白,为进一步研究VirB12蛋白的结构、功能及相关疫苗的研制奠定了基础。

Prokaryotic expression and purification of VirB12 protein of B.abortus

Objective To express the VirB12 protein of B.abortus in prokaryotic cells and purify the expressed product.Methods VirB12 gene was amplified from the genome of B.abortus by PCR and inserted into vector pET-30a(+).The constructed recombinant plasmid pETV12 was transformed to E.coli BL21(DE3)for expression under induction of IPTG.The expressed recombinant protein was purified by histidine-binding resin column chromatography and identify by SDSPAGE and Western blot.Results Recombinant plasmid pETV12 was constructed correctly as proved by restriction analysis and sequencing.The expressed recombinant protein,with a relative molecular mass of about 22 000,reached a purity of 94% after purification,was recognized by rabbit immune sera against B.abortus.Conclusion VirB12 protein of B.abortus was successfully expressed in prokaryotic cells and purified,which laid a foundation of further study on structure and function of VirB12 protein and development of the relevant vaccines.