非洲猪瘟病毒C129R蛋白与E165R蛋白的可溶性表达、胞外酶活力测定与结构预测分析  

Soluble expression,extracellular enzyme activity and structure prediction of C129R and E165R proteins of African swine fever virus

作　　者：王彦伟 岳亚男 张素玲 李欢欢 陈赵媛 刘月月 逄文强 田克恭 WANG Yanwei;YUE Ya'nan;ZHANG Suling;LI Huanhuan;CHEN-ZHAO Yuan;LIU Yueyue;PANG Wenqiang;TIAN Kegong(National Research Center for Veterinary Medicine,Luoyang 471000,China;Pulike Bioengineering Co.,Ltd.,Luoyang 471000,China)

机构地区：[1]国家兽用药品工程技术研究中心,河南洛阳471000 [2]普莱柯生物工程股份有限公司,河南洛阳471000

出　　处：《畜牧与兽医》2024年第7期52-57,共6页Animal Husbandry & Veterinary Medicine

基　　金：洛阳市重大科技专项(1901029A)。

摘　　要：C129R和E165R分别是非洲猪瘟病毒(ASFV)基因组编码的一种Mn依赖的超氧化物歧化酶和尿嘧啶脱氧核糖核苷三磷酸酶,2种酶均参与ASFV的复制过程。通过研究ASFV C129R蛋白与E165R蛋白的功能,以指导非洲猪瘟的诊断及免疫预防研究。对ASFV SY-18株的C129R和E165R基因序列进行密码子优化后,利用大肠杆菌进行表达,纯化后获得重组蛋白,通过Western blot鉴定其反应性,并对其胞外酶活力进行测定,对C129R蛋白的结构预测结果进行分析。结果:实现了C129R蛋白与E165R蛋白的可溶性表达,重组蛋白分子量分别约为15 kDa和18 kDa, Western blot显示C129R与E165R蛋白均能与ASFV阳性血清产生特异性反应;胞外酶活力测定显示,C129R与E165R蛋白在胞外均有酶学活性,比酶活力分别为(60.2±3.8) U/mg和(32.6±2.4) U/mg;蛋白质结构预测结果显示,C129R为双结构域蛋白,主要由N端α螺旋结构域和C末端α/β结构域组成,C129R蛋白N端α螺旋结构域和C末端α/β结构域的交界处可能是其主要活性位点,序列保守的β-折叠桶(K_(32)~A_(101))可能在其发挥功能时起重要作用。C129R is a Mn dependent superoxide dismutase encoded by the African swine fever virus(ASFV)genome,and E165R is a uridine deoxyribonucleoside triphosphate encoded by the ASFV genome,both of which are involved in the replication of ASFV.In order to study the extracellular functions of the C129R protein and E165R protein of ASFV,and to guide the diagnostic method and immunoprophylactic research of ASFV.In order to detect the functions of the C129R protein and E165R protein of African swine fever virus(ASFV),and to guide the diagnostic method for and immunoprophylactic research on ASFV.Codon optimization was executed on the sequences of C129R and E165R of ASFV SY-18,and the recombinant protein were expressed in Escherichia coli and purified.Then,the reactions of C129R and E165R were identified using Western blot and their extracellular enzyme activity was measured.Finally,the structure of C129R was predicted and analyzed,and soluble expression of the C129R and E165R proteins was achieved,with recombinant protein molecular weights of approximately 15 kDa and approximately 18 kDa,respectively.Western blot showed that C129R protein and E165R could produce specific reactions with ASFV positive serum.The determination of extracellular enzyme activity showed that both C129R and E165R had extracellular enzymatic activity,with specific enzyme activity of(60.2±3.8)U/mg and(32.6±2.4)U/mg,respectively.The structural prediction result of C129R showed that it was a dual domain protein,mainly composed of the N-terminalαdomain and C-Terminalα/βdomain.The junction of the C129R N-terminalαdomain and C-Terminalα/βdomain might be the main active site,and conservativeβbucket(K_(32)-A_(101))might play an important role in its function.This study laid a foundation for the diagnosis and immunoprophylaxis of ASFV.

关 键 词：非洲猪瘟病毒 C129R蛋白 E165R蛋白 可溶性表达 酶学活性 结构预测 

分 类 号：S852.65[农业科学—基础兽医学]