非洲猪瘟病毒CP204L基因的生物信息学分析、载体构建及蛋白表达

Bioinformatics Analysis,Vector Construction and Protein Expression of African Swine Fever Virus CP204L Gene

【目的】非洲猪瘟病(African swine fever,ASF)是一种由非洲猪瘟病毒(African swine fever virus,ASFV)导致的,能引起猪高度致死的急性传染病。采用生物学手段制备检测试剂能更快、更有效地监测疫情,对ASF疫情的防控尤为重要。【方法】根据ASFV的CP204L基因CDS区序列设计特异性引物并进行PCR扩增,将目的基因片段与p EASY-T5连接构建克隆载体,利用生物信息学软件对蛋白进行生物信息学分析,用T4连接酶构建重组载体pET-32a(+)-CP204L,将成功构建的重组表达载体质粒转入感受态Rosetta(DE3)中,用1 mmol/L IPTG、37℃诱导表达8 h后进行SDS-PAGE电泳鉴定蛋白表达。【结果】生物信息学分析结果表明,P30蛋白是一种主要由无规卷曲α螺旋和β折叠组成的亲水和稳定蛋白,PCR扩增及测序比对结果证明成功构建了重组表达载体,SDS-PAGE电泳验证了在24.3 ku处有潜在的P30蛋白表达。【结论】通过生物信息学软件在分子层面分析了P30蛋白的理化性质及蛋白结构,P30蛋白的表达为制备ASFV血清学诊断试剂及诊断技术奠定了基础。

【Objective】Caused by African swine fever virus(ASFV),African swine fever(ASF)is a highly lethal and acute infectious disease to pigs.The preparation of detection reagent by biological means can monitor the epidemic situation more quickly and effectively,which is particularly important for the prevention and control of the epidemic situation of ASF.【Method】Specific primers were designed according to the CP204L gene CDS region sequence of ASFV and PCR amplification was conducted.The target gene fragment was connected with p EASY-T5 clone vector.The bioinformatics software was used to analyze the protein bioinformatics.The recombinant vector pET-32a(+)-CP204L was constructed with T4 ligase.A successfully constructed recombinant expression vector plasmid was transferred into the receptive Rosetta(DE3).Protein expression was identified by SDS-PAGE electrophoresis after induced expression for 8 h with 1 mmol/L IPTG at 37℃.【Result】Bioinformatics analysis showed that P30 protein was a hydrophilic and stable protein.Bioinformatics analysis showed that P30 protein was a hydrophilic and stable protein composed of random coiledα-helix andβ-folding.PCR amplification and sequencing comparison results proved that the recombinant expression vector was successfully constructed.SDS-PAGE electrophoresis confirmed the potential expression of P30 protein at 24.3 ku.【Conclusion】The physical and chemical properties and protein structure of P30 protein were analyzed by bioinformatics software at the molecular level.The expression of P30 protein laid a foundation for the preparation of ASFV serological diagnostic reagents and diagnostic techniques.