摘要
目的:构建人血管能抑素canstatin基因原核表达载体,表达并纯化出其重组蛋白。方法:从人胎盘组织中提取总RNA,经逆转录-聚合酶链式反应(RT-PCR)扩增canstatin cDNA,克隆进质粒pUCm-T,转化E.coliDH5α,测序正确后,酶切目的基因片段,插入质粒pET-22b(+),转化E.coliBL21,IPTG诱导蛋白表达,N i-NTA柱亲和层析纯化重组蛋白。结果:电泳证实RT-PCR扩增产物与预期目的基因canstatin长度一致。RT-PCR纯化产物与载体pUCm-T连接后,经蓝白斑筛选,挑出6个白色菌落做酶切鉴定。其中一个菌落被证实为阳性克隆,测定其基因序列,结果显示与Genbank公布的canstatin基因序列完全一致。将canstatin cDNA,插入质粒pET-22b(+),转化E.coliBL21,培养过夜后,挑选7个白色菌落做酶切鉴定,证实均为阳性克隆。IPTG诱导其中一个克隆表达蛋白,SDS-PAGE电泳分析表明,在相对分子质量24 000左右出现新的蛋白表达条带,诱导1、2、3、4 h后,表达蛋白分别占菌体总蛋白的18.2%、18.8%、23.0%和23.4%。将诱导3 h的菌体超声破碎后,N i柱亲和层析,125和250 mmol/L咪唑洗脱液SDS-PAGE电泳显示出清晰的单一条带。结论:成功构建人canstatin基因原核表达载体,表达并纯化出canstatin重组蛋白,为深入研究其临床应用打下基础。
Objective ： To construct prokaryotic expression vector of human canstatin gene, express and purify its recombinant protein. Methods： The total RNA was extracted from human placenta tissues. The canstatin gene fragment was amplified from the total RNA by RT-PCR. The resulting product was cloned into pUCm-T vector and was sequenced. Then the confirmed canstatin cDNA was cloned into plasmid pET-22b（ ＋ ） and transformed into E. coli BI221 where it was induced to express proteins by isopropyl-l-thio-b-Dgalactopyranoside （IPTG）. The expression of protein was analyzed through SDS-PAGE. Cells induced 3 hours by IPTG were harvested, sonicated briefly and the proteins were purified through affinity chromatography. Results： The target sequences were specifically amplified through RT-PCR, cloned into pUCm-T vector, and then transformed into E. coli DH5α. Six white colonies were selected and cut by BamH Ⅰ and Hind Ⅲ separately. The plasmids of one white colony showed positive results and was sequenced, demonstrating to be the same as that of canstatin gene in GenBank. Then canstatin cDNA was cut down from pUCm-T, ligated into the vector pET-22b （ ＋ ） and then transformed into E. coli BL21. After an overnight incubation, seven white colonies were picked and showed positive results after digested by both BamH Ⅰ and Hind Ⅲ. After IPTG induction of one positive colony, a new protein band about Mr 24 000 showed on SDS-PAGE. The percent expressed product over total bacterial proteins after 1, 2, 3, and 4 hours of induction was 18.2% , 18.8% , 23.0% and 23.4% respectively estimated by densitometry. After affinity chromatography, SDS-PAGE showed only one clear band existed in 125 mmol/L or 250 mmol/L imidizone elution. Conclusion ： The prokaryotic expression vector of human canstatin gene has been successfully constructed. Further more, purified recombinant proteins are obtained through affinity chromatography, laying foundation for further study of its clinical use.
出处
《医学研究生学报》
CAS
2005年第11期981-985,共5页
Journal of Medical Postgraduates
基金
上海市科委基础研究重大课题基金资助项目(批准号:03JC14007)
