PCR产物靶向克隆法构建pET15b-CAT及His-tag-CAT融合蛋白的表达与纯化

目的:将人过氧化氢酶(Catalase,CAT)cDNA的PCR扩增产物定向克隆到pET15b载体上以构建重组质粒pET15b-CAT,并表达和纯化出His-tag-CAT融合蛋白。方法:扩增目的基因的PCR引物的5′端加上与线性化载体同源的碱基序列,以使PCR的扩增产物两端分别带上15个和线性化载体两端同源的碱基序列。以pZeoSV2(+)-CAT为模板进行靶向克隆所需的人CAT cDNA扩增。将纯化的人CAT cDNAPCR产物与经XhoⅠ和BamH Ⅰ酶切的线性化载体pET15b以6∶1摩尔数之比混合于含重组酶的反应液中,25℃反应30min,将PCR产物定向克隆于目标载体pET15b上,获得重组质粒pET15b-CAT。重组质粒经PCR,XhoⅠ酶切和DNA测序鉴定。pET15b-CAT转化BL21(DE3)宿主菌,用IPTG诱导表达出His-tag-CAT融合蛋白,采用Ni2+-NTA树脂进行亲和层析。结果:人CAT cDNA的PCR扩增产物与经XhoⅠ和BamHⅠ酶切的线性化载体pET15b发生了同源重组反应,经鉴定采用PCR产物靶向克隆法成功构建了pET15b-CAT。SDS-PAGE和Western blot结果表明,转化了pET15b-CAT的宿主菌表达出了His-tag-CAT融和蛋白,经Ni2+-NTA树脂亲和层析得到了纯化的His-tag-CAT,并在体外检测到其具有特异的过氧化氢酶的活性,活性值为80.23U/g。结论:采用PCR产物靶向克隆法成功地构建了pET15b-CAT,并表达和纯化出了His-tag-CAT融和蛋白,为采用外源性CAT防治与氧化应激损伤相关性疾病奠定了基础。

The Construction of Prokaryotic Expression Vector pET15b-CAT with PCR Cloning Reaction and Expression and Purification of His-tag-CAT Fusion Protein

Objective To clone human catalase (CAT) cDNA PCR product into pET15b-CAT to generate recombinant plasmid pET15b-CAT and express and purify His-tag-CAT fusion protein. Methods A pair of PCR primers having 15 bases of homology with sequences flanking the desired site of insertion in the cloning vector were designed. The plasmid pZeoSV2(+)-CAT was used as the template for amplification of human CAT cDNA for PCR cloning. The purified human CAT cDNA and the linearized pET15b with XhoⅠ and BamHⅠ digestion was mixed together at a molar ratio of 6:1 within the tube containing recombinant enzyme in total volume 20μL and incubated at 25 ℃ for 30 minutes,then transformed the reaction product into competent DH5α. The prokaryotic expression vector was identified with PCR,XhoⅠ digestion and DNA sequencing. The recombinant plasmid pET15b-CAT was transformed into BL21(DE3) host bacteria which was induced with IPTG. The recombinant protein His-tag-CAT was purified with affinity chromatography on a Ni2+-NTA-resin column. Results Homologous recombination occurred between PCR product human CAT cDNA and linearized pET15b. Human CAT cDNA was successfully inserted into pET15b to generate pET15b-CAT. SDS-PAGE and Western blot demonstrated successful expression and purification of His-tag-CAT fusion protein with specific activity of 80.23 U/g. Conclusion The prokaryotic expression vector pET15b-CAT has been constructed successfully with PCR cloning reaction. The successful expression and purification of His-tag-CAT fusion protein provides a basis for prevention and therapy of various disorders related to oxidative stress.