胰蛋白酶抑制剂KSTI3基因新型真核表达载体的构建及其在盐藻中的表达  

Construction of Trypsin Inhibitor KSTI3 Gene New Eukaryotic Expression System and Expression in Dunaliella salina

作　　者：徐文琦[1] 柴晓杰[1] 张婷[1] 代靖宇[1] 张晓琳[1] 

机构地区：[1]大连海洋大学海洋生物资源恢复与生境修复重点实验室,大连116023

出　　处：《中国生物工程杂志》2011年第8期29-34,共6页China Biotechnology

基　　金：辽宁省教育厅资助项目(2008T023)

摘　　要：应用PCR技术扩增Nos基因、CaMV35S启动子片段和氯霉素抗性基因Cat,并与胰蛋白酶抑制剂KSTI3基因连接,构建真核表达载体pMDCKN-Cat。DNA序列分析结果表明:表达载体中的胰蛋白酶抑制剂KSTI3基因、启动子CaMV35S、终止子Nos和氯霉素抗性基因Cat与已知序列完全一致。采用LiAc/PEG介导法将质粒pMDCKN-Cat转化至盐藻细胞中,通过氯霉素抗性基因筛选和PCR鉴定获得转基因盐藻细胞。经Western blotting检测,在硝酸纤维素膜上出现清晰的条带,分子量为20.1kDa,证明胰蛋白酶抑制剂KSTI3基因在盐藻中得到成功表达。To construct cloning vector pMDNos,terminator Nos was amplified from the template pBI121 plasmid by PCR.pMDKN vector was formed by combination between pMDNos fragment and KSTI3 from plasmid pMDKSTI3 by restriction enzyme digestion.Finally,promoter CaMV35S and chloramphenicol resistance gene Cat cloned from pCAMBIA2201 were all introduced to the vector pMDKN.DNA sequencing result showed that KSTI3 gene,promoter CaMV35S,terminator Nos and Cat gene were completely consistent with the original sequence,so new eukaryotic expression vector pMDCKN-Cat was successfully constructed.pMDCKN-Cat was transformed into Dunaliella salina（D.salina）with LiAc/PEG method,and recombinant D.salina was selected under chloramphenicol pressure and by PCR detection.Western blotting ultimately showed that a clear strip with the molecular weight of 20.1kDa appeared on the nitrocellulose membrane,and proved that trypsin inhibitor KSTI3 was expressed in D.salina cells.

关 键 词：胰蛋白酶抑制剂KST13 基因 盐藻 LiAc/PEG介导法 真核表达载体 

分 类 号：Q786[生物学—分子生物学]