非洲猪瘟病毒K196R和A240L蛋白的可溶性表达及酶活力分析

Soluble Expression and Enzyme Activity Analysis of African Swine Fever Virus K196R and A240L Proteins

旨在提供可用候选抗原蛋白,用于非洲猪瘟病毒(African swine fever virus,ASFV)的诊断及免疫预防研究。将ASFV SY18株K196R和A240L蛋白的编码基因进行密码子优化,并在其N端添加促溶标签进行融合表达;TEV酶切后利用亲和层析纯化目的蛋白,并通过Western blot和酶活检测试剂盒对其反应性和体外酶活力进行鉴定。结果显示,K196R和A240L蛋白分别通过添加SUMO和MBP标签实现了可溶性表达,标签去除后蛋白大小分别为22 kD和27 kD左右;Western blot结果显示,K196R和A240L蛋白均能够与ASF阳性血清发生较好的特异性反应;酶活检测结果表明,二者均具有一定的体外酶催化活性。利用大肠杆菌表达的ASFV K196R和A240L可溶性蛋白,具有较好的反应性和一定的体外酶催化活性,可为ASFV的诊断及蛋白的相关功能研究提供一定的依据。

The objective of this work is to provide effective antigens for the clinical diagnosis and immunoprophylaxis of African swine fever virus(ASFV).K196R and A240L coding sequences derived from ASFV SY18 were executed codon optimization and co-expression with soluble-tag in their N terminals.The target proteins were purified by affinity chromatography after TEV digestion,and their reactivity and enzymatic activity were evaluated through Western blot and enzyme activity detection kit,respectively.As results,the soluble expressions of K196R and A240L proteins were obtained by severally fusing with SUMO and MBP tag,and the size was about 22 kD and 27 kD after tag removed.Western blot results showed both K196R and A240L proteins well specifically reacted with the ASF positive serum.Enzyme activity detection results demonstrated the K196R and A240L proteins all presented definite enzyme catalytic activity in vitro.In conclusion,the soluble K196R and A240L proteins primarily expressed in Escherichia coli all show better reactivity and certain enzymatic activity in vitro,which will be the basis for the ASFV clinical diagnosis and protein relevant functions investigation.