家蝇溶菌酶MDLZM基因克隆、表达和序列分析  

Cloning expression and sequence analysis of Musca domestica lysozyme

作　　者：彭传林[1,2] 魏川川[1] 吴建伟[1] 修江帆[1] 王宇[1,3] 

机构地区：[1]贵阳医学院寄生虫学教研室,贵州贵阳550004 [2]皖北矿务局煤电集团总医院普外科 [3]贵州省疾病预防控制中心

出　　处：《中国公共卫生》2015年第6期764-766,共3页Chinese Journal of Public Health

基　　金：国家自然科学基金(81360254);国家科技支撑计划课题(2011BAC06B12)

摘　　要：目的探讨家蝇溶菌酶(MDLZM)基因及编码的蛋白质结构和特征。方法利用美国国家生物技术信息中心和瑞士生物信息学研究所的蛋白分析专家系统中有关基因和蛋白序列的分析工具,结合其他生物信息学分析软件包,从Gen Bank上家蝇基因组序列识别出编码MDLZM的基因,分析预测该蛋白质的结构功能;设计引物PCR扩增MDLZM基因,将其克隆到原核表达质粒PEASY-E1中,重组质粒在大肠杆菌中的表达产物用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定。结果 MDLZM基因序列最大开放阅读框(ORF)为435 bp,编码145个氨基酸,理论分子量为15 958.4 Da,等电点为8.65;构建的重组质粒经PCR、双酶切及测序鉴定确为MDLZM基因;SDS-PAGE分析结果显示,重组质粒可在大肠杆菌Origmi B/DE3中表达,表达产物纯化后得到的融合蛋白相对分子质量约为15958.4 Da,诱导18 h蛋白表达量最高,SDS-PAGE鉴定得到的蛋白条带与目的蛋白大小相符。结论成功构建PEASY-E1-MDLZM重组原核表达质粒并表达出融合MDLZM蛋白。Objective To analyze structural characteristics of the protein from Musca domestica lysozyme （MDLZM） by bioinformatics and to clone and express the novel gene of MDLZM for the study of the recombinant pro- tein. Methods Using bioinformatics software package and tools of bioinformatics at web sites of National Center for Bi- otechnology Information（ NCBI）, the Expert Protein Analysis System （ExPaSy）, and combining other bioinformatics software packages, the full-length gene encoding MDLZM protein from the Musca domestica genome library of GenBank was identified and then the characteristics of the deduced proteins was analyzed. The genes encoding MDLZM were am- plified by PCR, and cloned into a prokaryotic expression vector PEASY-E1. The recombinant plasmids were transformed into E. coli OrigmiB/DE3 and followed by the expression of the proteins induced by isopropyl-beta-D-thiogalactopyrano- side（ IPTG）. The recombinant proteins were purified with Ni 2 ＋ -charged imino diacetate（Ni-IDA） affinity chromatogra- phy, and detected with sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE）. Results MDLZM gene sequence contains an open reading frame of 435 bp and encodes 145 amino acids with a predicted molecular weight of 15 958.4 Da and a isoelectric point of 8.65. PCR. Double enzyme digestion and DNA sequencing confirmed that the re- combinant expression plasmid was successfully constructed. With SDS-PAGE, the recombinant plasmid PEASY-E1- MDLZM was expressed and purified in OrigmiB/DE3, and the molecular weight of 15 958.4 Da was noted in MDLZM protein bands. The highest amount of protein expression was generated 18 hours after the induction. The size of protein obtained by SDS-PAGE method was consistent with that of the target protein. Conclusion A novel gene coding MDLZM was cloned, expresed, and purified successfully.

关 键 词：家蝇 家蝇溶菌酶(MDLZM) 克隆 表达 序列分析 

分 类 号：R114[医药卫生—卫生毒理学]