家蝇抗真菌肽-溶菌酶基因的克隆、表达及序列分析  

Cloning,Expression and Sequence Analysis of Musca domestica Antifungal Peptide-1 and Musca domestica Lysozyme

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

机构地区：[1]贵阳医学院寄生虫学教研室,贵阳550004 [2]皖北煤电集团总医院,宿州234000 [3]贵州省疾病预防控制中心,贵阳550004

出　　处：《生物技术通报》2015年第5期200-205,共6页Biotechnology Bulletin

基　　金：国家科技支撑计划(2011BAC06B12);国家自然科学基金项目(81360254);贵州省科学技术基金项目(黔科合J字[2012]2038号)

摘　　要：旨在对家蝇抗真菌肽MAF-1-溶菌酶(LZM)基因进行生物信息学分析,并进行融合基因MAF-1-LMZ的克隆和表达分析。从Gen Bank获得家蝇抗真菌肽MAF-1和溶菌酶LZM的编码序列,分析和预测这两种蛋白质的结构和功能。PCR扩增融合蛋白质抗真菌肽-溶菌酶的基因MAF-1-LMZ,将其克隆到原核表达载体p ET-28a中,重组质粒p ET-28a-MAF-1-LMZ在大肠杆菌Origmi B/DE3中经用IPTG诱导表达,表达产物MAF-1-LMZ通过SDS-PAGE电泳进行鉴定,采用小试管法倍比稀释法进行活性验证。结果显示,融合蛋白质MAF-1-LMZ序列的ORF为969 bp,编码322个氨基酸残基,理论分子量为35 468.6 Da,等电点为8.31,在大肠杆菌Origmi B/DE3中得到成功表达。其纯化后的目的蛋白具有抗真菌活性。The aim of this study is to have bioinformatics analysis of Musca domesitca antifungal peptide-1 （ MAF-1 ） and lysozymeb （ LMZ ） , also clone fused MAF-1-LMZ gene and have expression analysis of it. The encoding sequences of MAF-1 and LMZ were fetched from GenBank, and the structures and functions of＇2 proteins were analyzed and predicted. The gene MAF-1-LMZ was amplified by polymerase chain reaction （ PCR ） , then was ligated into pET 28a and transformed into E. coli Origmi （ DE3 ） competent cell, and induced with [PTG. The fusion protein MAF-1-LMZ in the expression vector was analyzed by SDS-PAGE. The activity of the protein was validated by methods of small test tube and doubling dilution. The open reading frame of the MAF-1-LMZ was 969 bp, encoding a putative protein consisting of 322 amino acids with a predicted molecular weight of 35 468.6 Da and pI of 8.31, indicating the gene expressed in E. coil Origmi （ DE3 ） successfully. The purified target protein had the antifungal activity.

关 键 词：家蝇 抗真菌肽-溶菌酶 重组表达 序列分析 

分 类 号：Q78[生物学—分子生物学]