Lrrc10蛋白表达载体的构建

Construction of Lrrc10 Protein Expression Vector

为了构建重组质粒pET-Lrrc10和Lrrc10蛋白表达,利用Nco Ⅰ和BamH Ⅰ双酶切载体pMD18-T-Lrrc10,回收目的片段与经同样酶切后的表达载体pET-30a(+),转入E.coli strainDH5α,菌落PCR筛选阳性菌落,提取阳性菌质粒并进行PCR和酶切鉴定;将重组子转入E.coli strain BL21(DE3),于37℃振荡培养,用1 mmol/L IPTG诱导目的蛋白的表达。结果表明,成功构建了融合表达载体并命名为pET-Lrrc10。转入E.coli strain BL21(DE3)进行目的蛋白的表达,获得与预期分子量大小相一致的融合表达蛋白Lrrc10;Lrrc10蛋白以包涵体形式存在于宿主菌中,且IPTG诱导4小时,目的蛋白表达量最大。

To construct pET-Lrrcl0 and Express protein of Lrrcl0, pMD18-T-Lrrcl0 was digested by BamH I and Nco I to get Lrrcl0, which was ligated with pET-30a（ ＋ ） digested by BamH I and Nco I. Then the recombinant was transformed into E. coli strain DH5ct. Positive strains were identified by PCR to extract recombinants which were transformed into E. coli strain BL21 （DE3） to induce Lrrcl0 protein with IPTG at 37°C. The results show that the recombinant plamid was constructed successfully and was named as pET-Lrrcl0. After induced by IPTG, the E. coli strain BL21 （DE3） transformed by pETLrrcl0 expressed recombinant protein which was consistent with expected molecular weight. Lrrcl0 recombinant protein exis- ted as inclusion body and reached its expression peak after induced by IPTG for four hours.