结核分枝杆菌Rv3369基因的表达和纯化

Expression and purification of Rv3369 of Mycobacterium tuberculosis

在大肠杆菌中高效表达结核分枝杆菌Rv3369蛋白,获得纯化的重组蛋白rRv3369。通过聚合酶链反应(Polymerase chain reaction,PCR)扩增Rv3369基因;以质粒pET28a为表达载体,构建重组质粒,转化大肠杆菌BL21(DE3);以异丙基硫代半乳糖苷(IPTG)诱导表达目的蛋白,通过SDS-PAGE鉴定rRv3369在大肠杆菌中的表达,确定rRv3369在大肠杆菌中的表达形式;采用Ni-NTA His.Bind Resin来纯化重组蛋白。重组质粒pET28a-Rv3369中目的基因测序结果与报道序列相同。分子量约19.5kDa,表达量约占菌体总蛋白的18%,纯化后的重组蛋白样品经SDS-PAGE和激光密度扫描分析表明其纯度为90%以上,每100mL培养菌可获得1.56mg左右的重组蛋白。用亲和层析法纯化的重组蛋白纯度较好。

To obtain purified recombinant Rv3369 protein by means of expressing the Rv3369 protein of Mycobacterium tuberculosis in E. coli. The gene coding Rv3369 protein was amplified by polymerase chain reaction （PCR）, then was inserted into an expression vector pET28a to get recombinant plasmid. The recombinant plasmid was transformed into E. coli BL21 （DE3）and induced by IPTG. The expressed product was indentified by SDS-PAGE and purified by Ni- NTA His· Bind Resin. The sequence of Rv3369 in recombinant plasmid was the same with GenBank＇s report. The molecular mass of the product is 19.5kDa, which accounts for about 20% in the thalli proteins, and its purity is more than 90% analyzed by SDS-PAGE and laser scanning. The yield of recombinant protein is 1.56mg from 100mL of culture. Compared with other methods, purity of the recombinant protein is higher through affinity chromatography.