羊口疮病毒B2L和F1L截短融合基因原核表达产物的免疫原性分析  

ORFV B2L and F1L truncated fusion genes immunogenicity analysis of the prokaryotic expressed product

作　　者：鲜思美[1,2] 郑维豪 梁倩 张友 杨倩 顾庆林 包涛涛 青成欣 杜鹏 邱进杰 XIAN Si-mei;ZHENG Wei-hao;LIANG Qian;ZHANG You;YANG Qian;GU Qing-lin;BAO Tao-tao;QING Cheng-xin;DU Peng;QIU Jin-jie(College of Animal Science,Guizhou University,Guiyang 550025,China;Institute of Animal Disease Research of Guizhou,Guiyang 550000,China;Agriculture and Rural Bureau of Southeast Guizhou,Qiandongnan 556000,China;Chongqing Academy of Animal Sciences,Chongqing 402460,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]贵州省动物疫病研究所,贵州贵阳550000 [3]贵州省黔东南州农业农村局,贵州黔东南556000 [4]重庆市畜牧科学院,重庆402460

出　　处：《中国预防兽医学报》2023年第2期185-190,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：贵州省科技计划项目(黔科合基础-ZK[2023]一般106、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);重庆荣昌农牧高新技术产业研发专项(cstc2019ngzx0012)。

摘　　要：为分析羊口疮病毒(OrfV)B2L和F1L截短融合基因表达蛋白的反应原性,本研究分别选取B2L、F1L基因序列的主要抗原区域,采用重叠延伸PCR将二者融合后克隆至原核表达载体pET-32a中,构建重组质粒pET-32a-B2L-F1L,通过PCR、双酶切和测序鉴定,结果显示获得1080 bp的B2L-F1L融合基因目的片段,测序结果经比对分析正确无误,表明正确构建了重组表达质粒pET-32a-B2L-F1L。将pET-32a-B2L-F1L转化BL21(DE3)后经IPTG诱导,SDS-PAGE检测结果显示表达了39 ku的重组蛋白rB2L-F1L。采用Ni-IDA亲和层析方法纯化重组蛋白,经western blot鉴定,结果显示获得39 ku的单一特异性条带,表明rB2L-F1L的纯化效果和免疫原性均较好。采用rB2L-F1L纯化蛋白皮下多点注射免疫羔羊,采用ELISA方法检测免疫后不同时间羔羊血清中的OrfV抗体、IL-2、IFN-γ和IL-4细胞因子。结果显示,与生理盐水组相比,rB2L-F1L能够诱导羔羊产生OrfV特异性抗体,并可显著或极显著诱导羔羊分泌IL-2、IFN-γ和IL-4细胞因子(P<0.05、P<0.01);与弱毒活疫苗组比较,除在免疫后14 d~28 d时rB2L-F1L诱导羔羊产生的IL-2水平显著低于弱毒活疫苗组外(P<0.05),其他细胞因子在各时段两组均无显著差异(P>0.05)。本研究首次将OrfV优势抗原基因融合表达并分析了融合蛋白的免疫原性,为OrfV的检测技术和基因工程亚单位疫苗的研发奠定了良好基础。In order to analyze the immunogenicity of a fused proteins harboring the main antigen regions of B2L and F1L,The truncated B2L and F1L genes were obtained and cloned into the prokaryotic expression vector pET-32a by overlapping extension PCR,and the recombinant plasmid pET-32a-B2L-F1L was constructed and transformed into E.coli BL21(DE3)for protein expression.The results showed that the fragment of B2L-F1L fusion gene with a length of 1080bp was obtained and inserted into the pET-32a vector.After transfecting pET-32a-B2L-F1L into BL21(DE3),pET-32a-B2L-F1L/BL21(DE3)was induced with 0.2mmol/L IPTG,37℃for 5 hours.pET-32a-B2L-F1L/BL21(DE3)were induction condition was optimized to 0.2mmol/L IPTG at 37℃for 5 hours.The recombinant protein was then purified by Ni-IDA affinity chromatography.SDS-PAGE and western blot results showed that the target protein rB2L-F1L was 39ku.The purified rB2L-F1L protein was used to immunize the lamb subcutaneously at multiple points,and the OrfV antibody,IL-2,IFN-γand IL-4 cytokines in the serum of immunized lamb were detected by ELISA.The results showed that compared with the normal saline group,rB2L-F1L could induce the production of OrfV-specific antibodies,and also significantly induce the secretion of IL-2,IFN-γand IL-4 cytokines in lambs(P<0.05).Compared with the attenuated live vaccine group,except for the fact that the IL-2 secretion induced by rB2L-F1L was significantly lower than that of the attenuated live vaccine group on days 14 to 28,there was no difference in other cytokines between the two groups at each time period.In summary,the results demonstrated that the fused protein can induce the immune response in lamb and will provide a good basis for the detection technology of OrfV and the development of genetic engineering subunit vaccine.

关 键 词：羊口疮病毒 B2L基因 F1L基因 截短表达 免疫原性分析 

分 类 号：S852.65[农业科学—基础兽医学]