羊口疮病毒B2L、F1L截短融合基因的构建及生物信息学分析  

Construction and bioinformatics analysis of truncated fusion gene of Orf virus B2L and F1L

作　　者：付强 梁倩 鲜思美[2,3] 包涛涛 杨倩 顾庆林 郑维豪 杜鹏 青成欣 FU Qiang;LIANG Qian;XIAN Simei;BAO Taotao;YANG Qian;GU Qinglin;ZHENG Weihao;DU Peng;QING Chengxin(Liupanshui City Animal Husbandry and Aquaculture Development Center,Liupanshui 553001,China;College of Animal Science,Guizhou University,Guiyang 550025,China;Guizhou Institute of Animal Diseases,Guiyang 550025,China;Qiandongnan Prefecture Agriculture and Rural Affairs Bureau,Qiandongnan 556000,China)

机构地区：[1]六盘水市畜牧水产业发展中心,贵州六盘水553001 [2]贵州大学动物科学学院,贵阳550025 [3]贵州省动物疫病研究所,贵阳550025 [4]贵州省黔东南州农业农村局,贵州黔东南556000

出　　处：《黑龙江畜牧兽医》2022年第21期86-92,139,140,共9页Heilongjiang Animal Science And veterinary Medicine

基　　金：贵州省科技支撑计划项目(黔科合支撑[2018]2264);贵州省科学技术基金项目(黔科合基础[2019]1113号)。

摘　　要：为了构建羊口疮病毒(Orf virus,OrfV)B2L-F1L截短融合基因,并对该基因序列进行生物信息学分析,试验采用DNAStar生物信息学软件分别对B2L、F1L基因序列进行分析,截取B2L、F1L基因序列的主要抗原区域,分别设计引物,利用重叠延伸PCR(SOE-PCR)技术对截取的B2L、F1L基因主要抗原区域进行融合,并与pMD-18T载体连接,构建重组质粒pMD18-T-B2L-F1L,经PCR鉴定、双酶切和测序验证正确后,应用生物信息学在线软件对B2L-F1L截短基因编码的蛋白质进行信号肽、跨膜结构、亲疏水性、柔韧性、抗原指数、抗原决定簇、糖基化结合位点、磷酸化结合位点、二级结构和三级结构进行预测。结果表明:构建的B2L-F1L截短融合基因大小约为1080 bp,与预期目的片段大小相符;该蛋白有信号肽、无跨膜结构域,亲水性、抗原指数及柔韧性较好,有17个抗原决定簇,2个糖基化结合位点和28个磷酸化结合位点,二级结构中以α-螺旋和无规则卷曲占比较大。说明试验成功构建出羊口疮病毒B2L-F1L截短融合基因。In order to construct a truncated B2 L-F1 L fusion gene of Orf virus(OrfV), and to perform bioinformatics analysis on the gene sequence, DNAStar bioinformatics software was used to analyze the B2L and F1L gene sequences in the experiment. The main antigenic regions of B2L and F1L gene sequences were truncated;primers were designed respectively, and the main antigenic regions of the truncated B2L and F1L genes were fused by overlapping extension PCR(SOE-PCR) technology. It was ligated with pMD-18 T vector to construct recombinant plasmid pMD18-T-B2L-F1L. After PCR identification, double digestion and sequencing verification, the encoded protein was predicted by using bioinformatics online softwares to analyze signal peptide, transmembrane structure, hydrophobicity, flexibility, antigenic index, antigenic determinant, glycosylation binding sites, phosphorylation binding sites, and secondary and tertiary structures. The results showed that the size of the constructed B2L-F1L truncated fusion gene was about 1 080 bp, which was consistent with the expected size of the target fragment. The protein had a signal peptide, no transmembrane domain, good hydrophilicity, antigenic index and flexibility, which had 17 antigenic determinants, 2 glycosylation binding sites and 28 phosphorylation binding sites;α-helix and random coil accounted for a large proportion in secondary structure. The results indicated that the B2L-F1L truncated fusion gene was successfully constructed.

关 键 词：羊口疮病毒 B2L F1L SOE-PCR 生物信息学分析 

分 类 号：S855.3[农业科学—临床兽医学]