血清4型禽腺病毒fiber-2基因在昆虫细胞中的表达及其免疫原性分析  

Expression and immunogenicity analysis of serotype 4 fowl adenovirus fiber-2 in insect cells

作　　者：梅梅[1,2,3,4] 陆吉虎 张雪花[1,2,3,4] 唐应华 卢宇[1,2,3] 侯继波 MEI Mei;LU Ji-hu;ZHANG Xue-hua;TANG Ying-hua;LU Yu;HOU Ji-bo(Institute of Veterinary Immunology&Engineering,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;National Research Center of Engineering and Technology for Veterinary Biologicals,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Ministry of Education Key Lab for Avian Preventive Medicine/Key Laboratory of Jiangsu Preventive Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China)

机构地区：[1]江苏省农业科学院动物免疫工程研究所,江苏南京210014 [2]江苏省农业科学院国家兽用生物制品工程技术研究中心,江苏南京210014 [3]扬州大学兽医学院教育部禽类预防医学重点实验室/江苏省动物预防医学重点实验室,江苏扬州225009 [4]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：《中国预防兽医学报》2019年第12期1251-1255,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：教育部禽类预防医学/江苏省动物预防医学重点实验室开放课题(YF201801)。

摘　　要：为研究I群血清4型禽腺病毒(FAdV-4)fiber-2蛋白的免疫原性,本研究利用杆状病毒表达系统构建了含fiber-2基因的重组杆状病毒穿梭载体,将其转染Sf9细胞后获得重组杆状病毒rBA-fiber-2,并采用悬浮放大工艺表达重组fiber-2蛋白。通过间接免疫荧光试验(IFA)和western blot鉴定重组蛋白fiber-2的表达,并采用免疫琼扩试验(AGP)检测fiber-2蛋白效价。以fiber-2蛋白作为免疫原制备油乳剂疫苗免疫SPF鸡进行免疫效力试验,分别设fiber-2疫苗组、fiber-2疫苗配伍免疫增强剂CVCVA5组及生理盐水对照组。免疫后14 d、21 d和28 d采集SPF鸡外周血分离血清,利用AGP法检测其血清抗体水平。IFA结果显示fiber-2蛋白在昆虫细胞中获得表达;western blot结果显示该蛋白能够与FAdV-4阳性血清发生特异性免疫反应,蛋白分子量约62 ku;AGP结果显示fiber-2蛋白AGP效价达1:16。免疫后14 d两组实验组鸡均可检测到特异性琼扩抗体,免疫后28 d fiber-2疫苗组SPF鸡抗体AGP效价平均值可达1:24,配伍CVCVA5疫苗组SPF鸡抗体AGP效价平均值可达1:104。以上结果表明fiber-2蛋白具有一定的免疫原性,免疫后可诱导机体产生特异性抗体,本研究为进一步研制FAdV-4亚单位疫苗提供实验依据。In order to test the immunogenicity of the fiber-2 protein of group I avian adenovirus serotype 4(FAdV-4),in this study,the fiber-2 gene was amplified and cloned into baculovirus shuttle vector,and then transferred into Sf9 cells to obtain the recombinant baculovirus rBV-fiber-2.The fiber-2 protein was expressed in suspension amplification culture and identified by indirect immunofluorescence(IFA)and western blot,and its titer was detected by immuno-agarization assay(AGP).The fiber-2 protein was used as an immunogen to prepare oil emulsion vaccine for immunological efficacy test.The SPF chickens were divided into three groups.The first group of SPF chickens were immunized with fiber-2 oil emulsion vaccine.the second group were immunized with fiber-2 oil emulsion vaccine added with CVCVA5,and the third group were immunized with saline solution.The level of antibody against fiber-2 was detected at 14,21 and 28 days post vaccination(dpv)by AGP.The results of IFA showed that fiber-2 protein was successfully expressed in insect cells.The results of western blot indicated that the fiber-2 protein was about 62ku and could specifically react with the positive serum against FAdV-4.The AGP titer of fiber-2 was 1:16.The specific antibody against fiber-2 was detected in the first and second group of chickens at 14 dpv,and the average AGP titer of antibodies at 28 dpv in the first group was 1:24,while the titer was 1:104 in the second group.The results indicate that fiber-2 protein can induce the specific antibodies against fiber-2 after immunization.These findings provides an experimental basis for the further development of FAdV-4 subunit vaccine against.

关 键 词：血清4型禽腺病毒 纤突蛋白 表达 免疫原性 

分 类 号：S855.3[农业科学—临床兽医学]