家蝇溶菌酶MDLZM2基因的克隆、序列分析及原核表达

对家蝇溶菌酶(Musca domestica lysozyme,MDLZM2)基因进行克隆、序列分析,构建原核表达载体并在大肠杆菌中表达。从Gen Bank家蝇基因组中筛选获得MDLZM2基因。以该基因的序列设计引物,进行PCR扩增,测序分析获得该基因完整编码序列。运用生物信息学方法对该基因及其编码蛋白的基本理化性质、信号肽、二级结构、三级结构和保守结构域等方面进行预测和分析。构建p EASY-E1-MDLZM2重组质粒,转化到大肠杆菌BL21(DE3)p Lys S Chemically Competent Cell中进行诱导表达及纯化。结果表明MDLZM2基因ORF全长552 bp,编码183个氨基酸,理论分子量21.2 k Da;等电点为6.13,具有Lysozyme家族的蛋白保守结构域。成功构建重组原核表达p EASY-E1-MDLZM2并诱导表达、纯化重组蛋白,为进一步研究该蛋白的生物学及免疫学活性奠定了基础。

Cloning, sequence analysis and prokaryotic expression of Musca domestica lysozyme gene

To probe into the cloning of the Musca domestica lysozyme gene, and then construction of prokaryotic expression vector and expressed in Escherichia coli . Screening and isolating of Musca domestica lysozyme Gene from the Musca domestica genome library of GenBank .Analysissed the gene and encoded protein sequnece of lysozyme by the bioinformatics methods in the following aspects, such as general physical and chemical properties, signal peptide, secondary structure, tertiary structure and conserved domain.Then plasmid pEASY-E1-MDLZM2were transformed into Escherichia coli BL21(DE3)pLysS Chemically Competent Cell for expression and purification.The open reading frame of the MDLZM2 Gene was 552 bp that encded a putative protein with 183 amino acids .The protein, with predicted molecular weight 21.2 kDa and pI of 6.13 . It has the conserved lysozyme domain so belongs to lysozyme family . The result showed that the recombinant prokaryotic expression vector pEASY-E1-MDLZM2was successfully constructed and fusion protein was expressed and purified in E.coli , and established the basis for further researches in biology activity and immunologic activity .