摘要
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。
Objeotive 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 pETl5b with Xho I and BamH I digestion was mixed together at a molar ratio of6：l within the tube containing recombinant enzyme in total volume 20p.L and incubated at 25 ℃ for 30 minutes, then transformed the reaction product into competent DH5α. The prokaryotic expression vector was identified with PCR, Xho I 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 Ni^2＋ -NTA-resin column, Resuits Homologous recombination occurred between PCR product human CAT eDNA and linearized pET15b. Human CAT eDNA 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.
出处
《郧阳医学院学报》
2008年第3期193-198,F0002,共7页
Journal of Yunyang Medical College
基金
湖北省高等学校优秀中青年科技创新团队计划(No:T200811)
十堰市重大科技项目(No:2006030Z)
