家蝇几丁质酶基因的序列分析、克隆和诱导表达  

Sequence analysis,cloning and induced expression of chitinaseⅠ gene in housefly(Musca domestica)

作　　者：国果[1] 吴建伟[1] 吴沁怡[1] 付萍[1] 张勇[1] 

机构地区：[1]贵阳医学院寄生虫学教研室,贵阳550004

出　　处：《中国人兽共患病学报》2012年第6期570-573,共4页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(81160204;30960343);贵州省科技厅基金(黔科合[2010]3160);高校博士点学科专项科研基金(20105215120001)~~

摘　　要：目的对EST筛选得到的家蝇几丁质酶Ⅰ(MDCⅠ)基因进行序列分析,克隆其cDNA序列并在大肠杆菌中表达。方法采用EST测序技术从已构建的家蝇幼虫cDNA质粒文库中筛选到MDCⅠ基因,对其进行序列测定和分析。以该基因的cDNA文库质粒为模板,通过PCR方法对MDCⅠ基因进行扩增,以pET 28a(+)为载体构建重组质粒,再转化到表达宿主菌大肠杆菌BL21(DE3)中,IPTG诱导表达。表达产物通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行鉴定。结果 MDCⅠ基因全长751bp,编码251个氨基酸,理论分子量28.62家kDa;等电点5.78,有1个chitinase 18家族的活性位点。构建了具有正确基因序列的MDCⅠ重组表达质粒,重组蛋白在大肠杆菌BL21(DE3)中表达。结论 MDCⅠ基因可在原核表达系统中表达,为进一步研究该蛋白的生物学、免疫学活性奠定了基础。The aim of this study is to analyze and predict the structure and characteristics of genes and encoding proteins of MDCⅠ（Musca domestica chitinaseⅠ）,with the methods of cloning and expressing that gene.Sequence analysis revealed that the open reading frame of the cDNA encoded a 251-amino acid protein,which contained an NH2-terminal signal sequence（1-22）.The sequence identified with other insect chitinase was between 60% and 70%.The protein,with a predicted molecular weight of 28.62 kDa and pI of 5.78,had one active site of family 18 chitinase.The gene coding for MDCⅠwas amplified by polymerase chain reaction（PCR）,and then was inserted into vector pET 28a（＋） and induced with IPTG.The recombinant protein in the expression vector was analyzed by SDA-PAGE.Results indicated that the recombinant plasmid with the correct target gene was constructed,and the recombinant protein was expressed in E.coli BL21（DE3）.The target gene was cloned into the host bacterium and expressed correctly,and these results would establish the basis for further researches in biology and immunology of chitinase in housefly.

关 键 词：家蝇 几丁质酶 序列分析 基因克隆 表达 

分 类 号：R37[医药卫生—病原生物学]