扩展青霉碱性脂肪酶结构基因的克隆与原核表达  

Cloning and Prokaryotic Expression of Structural Gene Encoding Penicillium expansum Lipase

作　　者：卜秀娟[1] 孟宪梅[1,2] 柳增善[3] 代平 

机构地区：[1]吉林大学军需科技学院,长春130062 [2]吉林工商学院生物工程分院,长春130062 [3]吉林大学人兽共患病教育部重点实验室,长春130062 [4]长春长生基因药业股份有限公司,长春130012

出　　处：《中国生物制品学杂志》2010年第5期485-488,共4页Chinese Journal of Biologicals

基　　金：吉林省教育厅"十一五"科学技术研究项目(吉教科合字[2008]第273号)

摘　　要：目的克隆并原核表达扩展青霉碱性脂肪酶(PEL)结构基因。方法提取扩展青霉总RNA,经RT-PCR扩增PEL结构基因,克隆至原核表达载体pET-28a(+),构建重组表达质粒pET-28a-PEL,转化大肠杆菌BL21(DE3),经IPTG诱导表达,SDS-PAGE分析目的蛋白的表达形式及表达量,包涵体蛋白经变性、复性后,采用NaOH滴定法测定其酶活性。结果经RT-PCR扩增获得约780bp的PEL结构基因;所构建的重组表达质粒pET-28a-PEL经酶切及PCR鉴定正确;目的蛋白的相对分子质量约为28000,以包涵体形式表达,表达量约占菌体总蛋白的10%;发酵液及复性的包涵体蛋白酶活性可达12.44U/ml。结论已成功克隆并原核表达了PEL结构基因,为获得碱性脂肪酶高产基因工程菌株奠定了基础。Objective To clone the structural gene encoding Penicillium expansum lipase（PEL）and express in prokaryotic cells.Methods Total RNA was extracted from Penicillium expansum for amplification of PEL gene by RT-PCR.The amplified PEL gene was cloned into prokaryotic expression vector pET-28a（＋）,and the constructed recombinant plasmid pET-28a-PEL was transformed into E.coli BL21（DE3）for expression under induction of IPTG.The form and expression level of target protein were analyzed by SDS-PAGE.The expressed product in a form of inclusion body was de-naturalized and re-naturalized,then determined for enzyme activity by titration with sodium hydroxide.Results The structural gene at a length of 780 bp was amplified by RT-PCR.Both restriction analysis and sequencing proved that recombinant plasmid pET-28a-PEL was constructed correctly.The target protein,with a relative molecular mass of about 28 000,was expressed in a form of inclusion body,which contained about 10% of total somatic protein.Both the enzyme activities of inclusion body protein in fermentation liquid and after renaturation reached 12.44 U /ml.Conclusion The structural gene of PEL was successfully cloned and expressed,which laid a foundation of establishment of recombinant E.coli for production of PEL in a large quantity.

关 键 词：扩展青霉 碱性脂肪酶 结构基因 原核表达 

分 类 号：Q785[生物学—分子生物学] Q786