家蝇幼虫防御素基因MddⅠ的克隆与原核表达  

Cloning and prokaryotic expression of defensinⅠgene MddⅠin Musca domesticalarvae

作　　者：万玲[1] 傅小蒙 唐艳[1] 孙小宁[1] 裴志花[1] 刘树明[1] 马红霞[1,2] 

机构地区：[1]吉林农业大学动物科学技术学院,吉林长春130118 [2]吉林农业大学动物生产及产品质量安全教育部重点实验室,吉林长春130118

出　　处：《中国兽医科学》2013年第12期1280-1284,共5页Chinese Veterinary Science

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);吉林省世行贷款农产品质量安全项目(2011-Y05);吉林省科技发展计划项目(20111820,20120229)

摘　　要：以猪肺炎支原体诱导家蝇幼虫的cDNA为模板,通过cDNA末端快速扩增技术(RACE)对MddⅠ基因进行扩增,对扩增产物测序和分析。构建重组表达质粒pET-32a-MddⅠ,在大肠杆菌BL21(DE3)中用IPTG对其诱导表达,对表达产物进行SDS-PAGE分析。结果,MddⅠ基因全长475bp,其开放阅读框(ORF)276bp,编码91个氨基酸,编码蛋白的理论分子质量9.7ku,理论等电点(pI值)8.89,存在信号肽的剪切位点,亲水性较强且无明显跨膜区,α螺旋和不规则盘绕为其主要结构元件,β折叠散布在整个蛋白质中。具有Defensin-2家族保守区域,与GenBank中登录号为AY260152同源性最高,达78%。构建的重组表达质粒pET-32a-MddⅠ在大肠杆菌BL21(DE3)中表达出分子质量约为25ku的蛋白质,与预期大小一致。结果表明,MddⅠ基因具有完整的开放阅读框,为新的家蝇防御素基因;MddⅠ基因在原核表达系统中的表达为进一步研究表达产物的生物学活性和免疫学活性奠定了基础。The aim of this study is to clone,analyze and express gene o{ Mdd I （Musca domestica defensin I ） being screened from SSH library induced by Mycoplasma pneumonia. Using cDNA end rapid am- plification technology（RACE）,Mdd I gene＇s full-length cDNA was amplified. The open reading frame （ORE）of Mdd I gene was amplified by polymerase chain reaction（PCR） and sequenced. A recombinant ex pression plasmid was reconstructed using pET-32a, and the expression of the recombinant plasmid was transfected into Escherichia coli BL21（DE3） and then induced by IPTG. The recombinant protein was veri fied by SDS PAGE. In result,the full-length cDNA of Mdd I gene was 475 bp,in size,encoding a 91-amino acid protein with a theotetical molecular weight of 9.7 ku,pI of 8.89,strong hydrophilicity,and no transmembrane region. The a screw and irregular coil were predicted to be the main structural components, and β strands were distrubuted throughout Mdd I . These results revealed that Mdd I gene might be a new de- fense gene in Musca domestica with the complete open reading frame and was expressed successfully in Escherichia coli BL21（DE3）,which is a basis for further researches in the biological and immunologic activities of Mdd I

关 键 词：家蝇 防御素 序列分析 克隆 原核表达 

分 类 号：S852.743[农业科学—基础兽医学]