牛布鲁菌L7/L12蛋白的原核表达及纯化

Prokaryotic Expression and Purification of L7/L12 Protein of B.abortus

目的原核表达并纯化牛布鲁菌L7/L12蛋白。方法提取牛布鲁菌S19基因组DNA,PCR法扩增L7/L12基因,经测序正确后,克隆至pET-28a(+)载体,构建重组原核表达质粒pET-L7/L12,转化大肠杆菌BL21(DE3),IPTG诱导表达。SDS-PAGE分析目的蛋白的表达形式及表达量,经亲和层析纯化后,Western blot鉴定纯化产物。结果所构建的重组原核表达质粒pET-L7/L12经酶切鉴定表明构建正确。重组蛋白以包涵体形式表达,表达量占全菌总蛋白的33%;纯化的重组蛋白纯度可达94%,可与布鲁菌小鼠免疫血清发生特异反应。结论已成功表达并纯化了牛布鲁菌L7/L12蛋白,为建立高特异性的新型布鲁菌检测方法奠定了基础。

Objective To express the L7/L12 protein of B. abortus in prokaryotic ceils and purify the expressed product. Methods The genomic DNA of B. abortus S19 was extracted for amplification of L7 / L12 gene by PCR. The amplified gene was identified by sequencing and cloned into vector pET-28a （＋）. The constructed recombinant plasmid pET-L7/L12 was transformed to E. coli BL21 （DE3） for expression under induction of IPTG, The expressed product was analyzed for form and expression level by SDS-PAGE, then purified by affinity chromatography and identified by Western blot. Results Restriction analysis proved that recombinant plasmid pET-L7 / L12 was constructed correctly. The expressed product, in a form of inclusion body, contained 33% of total somatic protein, reached a purity of 94% after purification and showed specific reaction with murine sera against B. abortus. Conclusion The L7 / L12 protein of B. abortus was successfully expressed and purified, which laid a foundation of developing a novel method with high specificity for determination of B. abortus.