家蝇溶菌酶1基因的克隆、序列分析及原核表达  

Cloning,Sequence Analysis and Prokaryotic Expression of the Lysozyme 1 Gene from Musca domestica

作　　者：王梅梅[1] 万玲[2] 孔令聪[2] 裴志花[2] 刘树明[2] 马红霞[2,3] 

机构地区：[1]吉林农业大学生命科学学院,长春130118 [2]吉林农业大学动物科学科技学院,长春130118 [3]动物生产与产品质量安全教育部重点实验室(吉林农业大学),长春130118

出　　处：《中国兽药杂志》2014年第3期10-14,共5页Chinese Journal of Veterinary Drug

基　　金：国家自然科学基金资助项目(31140026);教育部新世纪优秀人才项目(NCET-10-0174)

摘　　要：采用cDNA末端快速扩增技术(RACE),对鸡致病性大肠杆菌诱导家蝇幼虫抑制性消减文库(SSH)中筛选得到家蝇溶菌酶1基因((Musca domestica lysozyme-1,MdL-1)进行扩增,测序分析得到一个全长为537 bp的cDNA片段,其开放阅读框(ORF)432 bp,与Genbank中登录号为AY344589.1基因同源性为96%。构建重组表达质粒pET-32a-MdL-1,转化大肠杆菌BL21(DE3),异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析显示,表达产物大小约为34 kD,与预期蛋白大小一致。结果表明,利用RACE技术克隆得到MdL-1全长基因并在大肠杆菌中获得了表达,为进一步研究该蛋白的生物学及免疫学活性奠定了基础。A partial cDNA sequence of lysozyme 1 gene from Musca domestica induced by pathogenic Escherichia coli of chicken was screened by SSH library of Musca domestica larvae. Its full-length cDNA（537 bp） was cloned by cDNA end rapid amplification technology （RACE）.The cDNA contains a 432 bp open reading frame （ORF）, the homology of this gene with the MdL1 from GenBank was up to 96%. The MdL1 gene was ligated into expression vector pET-32a（＋） and transformed into E.coli BL21 competent cell for expression .After induced by IPTG,the 34 kD of fusion protein was obtained followed by SDS-PAGE analysis.The results showed that MdL1 cloned by RACE can express successfully in Escherichia coli BL21 （DE3） and establish the basis for further researches in biology activity especially in enteropathogenic Escherichia coli and immunologic activity.

关 键 词：家蝇 家蝇溶菌酶1 克隆 序列分析 原核表达 

分 类 号：S852.743[农业科学—基础兽医学]