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目的对家蝇热休克蛋白20(HSP20)基因进行生物信息学分析,并进行克隆、表达研究。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HSP20的基因,分析预测该蛋白质的结构功能;设计引物PCR扩增HSP20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Origmi B/DE3中经用异丙硫代-β-D半乳糖苷(IPTG)诱导表达,表达产物用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDSPAGE)鉴定。结果 HSP20基因序列全长865bp,最大开放阅读框(ORF)为567bp,编码188个氨基酸,理论分子量为21443.2 Da,等电点为5.96;所构建的重组质粒经PCR、双酶切及测序鉴定成功;SDS-PAGE分析结果显示,重组质粒在Origmi B/DE3中表达并纯化,得到的融合蛋白相对分子质量约为21443.2 Da,诱导12 h蛋白表达量最高,SDS-PAGE法得到的蛋白条带与目的蛋白大小相符。结论成功构建PEASY-E1-HSP20重组原核表达质粒并表达出融合HSP20蛋白,为进一步研究该蛋白的功能奠定基础。
Objective To study the bioinformatics analysis of HSP20 gene in housefly and to clone and express HSP20 gene. 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 HSP20 gene was analyzed to predict the structure and function of the protein. PCR was used to amplify the HSP20 gene by PCR and cloned into prokaryotic expression plasmid PEASY-E1. The recombinant plasmid was transfected into E. coli Origmi B / DE3 with isopropylthio-β -D galactoside (IPTG) induced expression, the expression product was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). Results The sequence of HSP20 gene was 865bp in length with a 567bp open reading frame (ORF) encoding 188 amino acids with a theoretical molecular weight of 21443.2 Da and an isoelectric point of 5.96. The recombinant plasmid was successfully identified by PCR, double enzyme digestion and sequencing SDS-PAGE analysis showed that the recombinant plasmid was expressed and purified in Origmi B / DE3. The relative molecular mass of the fusion protein was about 21443.2 Da, and the protein expression was highest at 12 h. SDS- The size of the target protein match. Conclusion The recombinant prokaryotic expression plasmid PEASY-E1-HSP20 was successfully constructed and the fusion protein HSP20 was expressed, which laid the foundation for further study on the function of this protein.