C株猪瘟病毒NS3蛋白抗原表位分析及分段表达活性检测  

Analysis of Epitope of NS3 Protein of Classical Swine Fever Virus Strain C and Detection of Its Segmented Expression Activity

作　　者：丁国伟 李甜甜 徐萍 魏荣荣 李琛 王设市 潘晨 范娟 DING Guo-wei;LI Tian-tian;XU Ping(Yangzhou Youbang Biological Medicine Co.,Ltd.,Yangzhou,Jiangsu 225008)

机构地区：[1]扬州市扬州优邦生物药品有限公司,江苏扬州225008

出　　处：《安徽农业科学》2023年第18期115-120,共6页Journal of Anhui Agricultural Sciences

基　　金：扬州市科技计划项目(YZ2021098)。

摘　　要：以经典毒株C株的基因组为模板,测序后对NS3蛋白的二级结构进行分析,并对抗原表位进行预测,结果表明NS3的二级结构非常丰富并且存在丰富的B细胞抗原表位和T细胞抗原表位。截获3个不同的基因区域a、b和c,并设计相应的引物,分别扩增出约645、609和615 bp的靶基因,分别用pET-28a表达载体进行重组后,获得pET-28a-NS3a、pET-28a-NS3b与pET-28a-NS3c,用表达菌株BL21(DE3)进行表达,b段表达较高,可溶性表达较好,纯化后,可获得重组蛋白CSFV-His-NS3b, SDS-PAGE验证表达的重组蛋白大小约为27 kD,Western-Blot证实其具有良好的免疫原性,并且通过ELISA测得蛋白浓度约5 mg/mL,表明该蛋白特异性很强,可用于猪瘟诊断试剂盒的开发应用。Using the genome of classical virus strain C as a template,the secondary structure of NS3 protein was analyzed and the Epitope were predicted after sequencing.The results showed that NS3 had very rich secondary structure and rich B cell Epitope and T cell Epitope.Three different gene regions a,b,and c were intercepted,and corresponding primers were designed to amplify target genes of approximately 645,609,and 615 bp,respectively.After recombination with pET-28a expression vector,pET-28a-NS3a,pET-28a-NS3b,and pET-28a-NS3c were obtained.The expression strain BL21(DE3)was used for expression,with high expression in segment b and good soluble expression.After purification,a recombinant protein CSFV-His-NS3b was obtained.The size of the recombinant protein confirmed by SDS-PAGE was about 27 kD,and Western-Blot confirmed its good immunogenicity.The protein concentration measured by ELISA was about 5 mg/ml,indicating that the protein has strong specificity and can be used for the development and application of swine fever diagnostic kits.

关 键 词：NS3蛋白 表达 诊断 

分 类 号：S852.65[农业科学—基础兽医学]