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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。
Objective To express and purify the Brucella VirB12 protein in prokaryotic cells. Methods VirB12 gene was amplified by PCR from Brucella bovinum and inserted into pET-30a (+) vector to construct recombinant expression plasmid pETV12. The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced by IPTG. The expressed recombinant protein was purified by histidine-binding resin column and analyzed by SDS-PAGE and Western blot. Results The recombinant plasmid pETV12 was confirmed by double enzyme digestion and sequencing. The expressed recombinant protein had a relative molecular mass of about 22 000 and the purity of the purified recombinant protein was 94%. It could be specifically recognized by Brucella immunized rabbit serum. Conclusion Prokaryotic expression and purification of VirB12 protein of Brucella bovinum have laid the foundation for further study of the structure and function of VirB12 protein and the development of related vaccines.