人组织激肽释放酶6的原核表达载体的构建及表达

Construction and Expression of A Protokaryon Vecter Encoding Human Kallikrein 6

目的:构建人组织激肽释放酶6原核表达系统。方法:采用RT-PCR技术从人乳腺癌组织中扩增出KLK6基因片段,克隆至原核表达载体pET28b中,经酶切和序列测定后,转化至E.coli BL21,IPTG进行诱导表达,表达产物经SDS-PAGE检测,Ni-NTA亲和层析柱纯化,其纯化产物进行SDS-PAGE和Western-blot检测。结果:pET28b-KLK6原核表达载体构建成功,经IPTG诱导高效表达hK6蛋白,纯化后获得大量高纯度蛋白。结论:成功构建了KLK6的原核表达系统并在E.coli BL21中高效表达,获得高纯度的hK6融合蛋白。

Objective To construct the protokaryon expression system of human kallikrein 6.Methods The total RNA was extracted from the tissues of breast invasive ductal carcinomas.The KLK6 fragment was amplified by the RT-PCR method and cloned into the plasmid pET28b.After identified by restriction endonuclease digestion analysis and DNA sequencing,the pET28b-KLK6 was transformed into E.coli BL21 and induced with IPTG to express hK6.The expressed protein was detected by SDS-PAGE.After Ni-NTA affinity chromatography,the purification product was detected by SDS-PAGE and Western-blot technique.Results The recombinant protokaryon expression vector of pET28b-KLK6 was constructed successfully.The BL21 transfromed with the pET28b-KLK6 plasmid expressed a high level of the hK6 protein in the cytoplasm,then the high-purity hK6 was got by Ni-NTA affinity chromatography.Conclusion The prokaryotic expression system of human kallikrein 6 was successfully constructed in BL21,and the high purity protein was obtained.