裂谷热病毒eGn蛋白的制备及免疫原性验证  

Preparation and immunogenicity verification of eGn protein of Rift Valley fever virus

作　　者：韩秋雪 黄培 毕津豪[2,4] 金宏丽 赵永坤 焦翠翠 张胜男 徐胜男 张颖 曹增国 迟航[2] 王化磊 杨松涛 夏咸柱 HAN Qiu-xue;HUANG Pei;BI Jin-hao;JIN Hong-li;ZHAO Yong-kun;JIAO Cui-cui;ZHANG Shcng-nan;XU Sheng-nan;ZHANG Ying;CAO Zeng-guo;CHI Hang;WANG Hua-lei;YANG Song-tao;XIA Xian-zhu(College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China;Institute of Military Veterinary Medicine,Academy of Military Sciences,Academy of Military Medical Sciences,Changchun 130122,China;Key Laboratory of Zoonosis Research .Ministry of Education,College of Veterinary Medicine,Jilin University,Changchun 130062,China;College of Animal Science and Technology,Jilin Agricultural University .Changchun 130118,China;College of Wildlife Resources,Northeast Forestry University,Harbin 150040,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses,Yangzhou,Jiangsu 225009,China)

机构地区：[1]东北农业大学动物医学学院,黑龙江哈尔滨150030 [2]军事科学院军事医学研究院军事兽医研究所,吉林长春130122 [3]吉林大学动物医学学院,人兽共患病研究教育部重点实验室,吉林长春130062 [4]吉林农业大学动物科学技术学院,吉林长春130118 [5]东北林业大学野生动物资源学院,黑龙江哈尔滨150040 [6]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医学报》2019年第8期1506-1512,共7页Chinese Journal of Veterinary Science

基　　金：国家科技支撑计划基金资助项目(2015BAD12B04)

摘　　要：通过PCR扩增裂谷热病毒(RVFV) Gn蛋白胞外区(eGn)基因,连入原核表达载体pET-30a(+),构建重组质粒pET-30a(+)-RVFV-eGn。将重组质粒转化至感受态BL21(DE3),IPTG诱导蛋白表达,经HisTrapTMFF蛋白纯化柱纯化目的蛋白。将目的蛋白免疫BALB/c小鼠,分离小鼠脾细胞与小鼠骨髓瘤细胞融合,筛选分泌特异性抗体的杂交瘤细胞株,制备单克隆抗体,免疫荧光鉴定制备的单克隆抗体特性。结果显示,PCR扩增出1 287 bp的目的基因;构建的重组质粒转化BL21细胞后可有效表达目的蛋白,确定最佳表达条件为30℃,0.8 mmol/L IPTG诱导5 h,目的蛋白以包涵体形式表达;纯化后目的蛋白纯度为94.136%;制备的单克隆抗体可特异性识别哺乳动物细胞表达的Gn蛋白,表明RVFV eGn蛋白具有良好的免疫原性。研究结果为裂谷热新型疫苗和快速诊断试剂的研制奠定基础。The extracellular domain(eGn) gene of Rift Valley fever virus(RVFV) Gn protein was amplified by PCR and inserted into the prokaryotic expression vector pET-30 a(+) to construct the recombinant plasmid pET-30 a(+)-RVFV-eGn,which was transformed into competent BL21(DE3).Protein expression using IPTG was purified by HisTrapTM FF protein purification column for target protein.Immunized BALB/c mice using the purified protein,and the mouse spleen cells were isolated and fused with myeloma cells.The hybridoma cell lines secreting specific antibodies were screened to prepare monoclonal antibodies,and the characteristics of monoclonal antibodies were determined by immunofluorescence.The results showed that the 1 287 bp target gene was amplified by PCR,the constructed recombinant plasmid was successfully transformed into BL21 cells,and the target protein was effectively expressed.The optimal expression conditions were30℃,0.8 mmol/L IPTG induction for 5 h,and the target protein was in clusion form expression.The purity of the target protein after purification was 94.136%,and the prepared monoclonal antibody specifically recognized the Gn protein expressed by mammalian cells.It has been noted that the eGn protein of Rift Valley fever has good immunogenicity and laid a foundation for the development of new vaccines and rapid diagnostic reagents of Rift Valley fever.

关 键 词：裂谷热病毒 Gn胞外区(eGn) 单克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学] R535[农业科学—兽医学]