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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 investigate the structure and characteristics of housefly lysozyme (MDLZM) gene and its encoded protein. Methods Based on the analysis tools of gene and protein sequences in protein analysis expert system of National Institute of Biotechnology Information Center and Swiss Bioinformatics Institute, combined with other bioinformatics analysis software packages, the coding sequence of housefly in Gen Bank was identified MDLZM gene was analyzed to predict the structure and function of the protein. MDLZM gene was amplified by PCR and cloned into prokaryotic expression plasmid PEASY-E1. The recombinant plasmid was expressed in E.coli with sodium dodecyl sulfate- Polyacrylamide gel electrophoresis (SDS-PAGE) identification. Results The ORF of MDLZM gene was 435 bp, encoding a protein of 145 amino acids with a theoretical molecular weight of 15 958.4 Da and an isoelectric point of 8.65. The recombinant plasmid was identified as MDLZM by PCR, double enzyme digestion and sequencing SDS-PAGE analysis showed that the recombinant plasmid was expressed in Escherichia coli Origmi B / DE3. The relative molecular mass of the fusion protein was about 15958.4 Da after purification, and the highest level of protein was induced at 18 h after SDS-PAGE. The protein band and the size of the target protein. Conclusion PEASY-E1-MDLZM recombinant prokaryotic expression plasmid was successfully constructed and fused with MDLZM protein.