摘要
目的构建人类肝脏的cDNA文库,从中筛选hHGF基因并进行克隆与表达。方法从人类胎儿肝脏中快速分离提取出mRNA;将已提取出的mRNA构建成cDNA文库;从中筛选出hHGF(hepatocyte growth factor)基因后,进行克隆并测序确定;构建表达质粒pBV221-hHGF后转化大肠杆菌BL21(DE3α),以异丙基硫代-B-D-半乳糖苷(IPTG)诱导,收集菌体后提取蛋白样品并对其进行SDS-PAGE电泳及蛋白免疫印迹检测。结果成功地构建了人类肝脏的cDNA文库;经筛选得到约2200bp的hHGF基因;对其进行测序确认;成功地构建该基因的表达型质粒,并对表达蛋白进行蛋白免疫印迹检测,检测出其表达产生的蛋白质相对分子量在100000左右。结论人类肝脏cDNA文库成功地构建后可用于筛选获取完整的cDNA基因,从中筛选、克隆hHGF基因,并测序确认,进行表达蛋白质检测,结果正确,以上工作为进一步深入地进行与人类肝脏cDNA文库及hHGF相关的实验研究奠定了一定的基础。
Objective To construct the cDNA library of human foetus＇ liver, and screen hHGF （human hepatocyte growth factor） gene from it, then perform cloning and expressing of the gene. Methods The mRNA was separated from human foetus＇ liver, and constructed a library of it. After screening hHGF gene from it, the gene was cloned and checked by sequence determination. The plasmid pBV221-hHGF was recombinanted and transferred it into BL21 （DE3α） E.coli. The E.coli was induced by IPTG, and the expressed proteins were determined by SDS-PAGE electrophoresis and Western-blotting. Results The cDNA library of human liver was constructed successfully, and the hHGF gene about 2 200 bp was obtained in screening. The hHGF was confirmed by sequence determination, and expressed in E.coli. after recombinanting plasmid pBV221-hHGF. Its protein was verified by SDS-PAGE electrophoresis and Western-blotting, and its molecular weight was about 100 000. Conclusion The human liver cDNA library is constructed successfully. The intact cDNA gene can be screened from it. The cloning, sequencing and expressing of hHGF gene are successful. This work establishes a certain basis for further experiemental study on human liver cDNA library and hHGF genes.
出处
《生物医学工程与临床》
CAS
2006年第5期315-319,共5页
Biomedical Engineering and Clinical Medicine
基金
吉林省科委科研基金资助
