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当前位置: 首页 > 文章 > 犬瘟热病毒囊膜糖蛋白在杆状病毒-昆虫细胞系统中的表达及鉴定 畜牧兽医学报 2015,46 (6) 981-988
Position: Home > Articles > Expression and Identification of Glycoprotein of Canine Distemper Virus in the Baculovirus-Insect Cell System Chinese Journal of Animal and Veterinary Sciences 2015,46 (6) 981-988

犬瘟热病毒囊膜糖蛋白在杆状病毒-昆虫细胞系统中的表达及鉴定

作  者：
虞一聪;冯娜;闫飞虎;盖微微;王铁成;王化磊;郑学星;赵永坤;黄耕
单  位：
吉林农业大学动物科学技术学院;军事医学科学院军事兽医研究所
关键词：
犬瘟热病毒;融合蛋白;血凝素蛋白;杆状病毒-昆虫细胞系统
摘  要：
为表达具有天然构象的犬瘟热病毒(CDV)囊膜糖蛋白融合蛋白(F)和血凝素蛋白(H),本研究扩增小熊猫源CDV驯化致弱株LP的F、H基因,克隆至pFastBacTM1载体中,测序验证后转化至DH10BacTM感受态细胞,同源重组获得穿梭质粒rBacmid-F、rBacmid-H,将其分别转染Sf9细胞获得重组杆状病毒rpFB-F、rpFB-H,并将表达的重组融合蛋白(rF)和血凝素蛋白(rH)进行IFA和Western blot鉴定。以犬抗CDV高免血清对重组杆状病毒感染细胞进行IFA鉴定,在感染细胞的细胞膜上可见特异性荧光反应;以鼠抗F、H蛋白的主要抗原表位区多克隆抗体对重组杆状病毒感染细胞进行Western blot检测,可见相对分子质量为63和68ku左右的条带,分别为重组融合蛋白(rF)和血凝素蛋白(rH),大小与预期相符。两种囊膜糖蛋白在杆状病毒-昆虫细胞系统中均成功表达,且具有良好的反应原性。本研究为CDV病毒样颗粒疫苗的开发等工作奠定了基础。
译  名：
Expression and Identification of Glycoprotein of Canine Distemper Virus in the Baculovirus-Insect Cell System
作  者：
YU Yi-cong;FENG Na;YAN Fei-hu;GAI Wei-wei;WANG Tie-cheng;WANG Hua-lei;ZHENG Xue-xing;ZHAO Yong-kun;HUANG Geng;YANG Song-tao;GAO Yu-wei;XIA Xian-zhu;College of Animal Science and Technology,Jilin Agricultural University;Key Laboratory of Zoonosis Prevention and Control of Jilin Province,Military Veterinary Institute of the Academy of Military Medical Sciences;Changchun Veterinary Research Institute,Chinese Academy of Agricultural Sciences;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis;
关键词：
canine distemper virus;;fusion protein;;attachment glycoprotein;;baculovirus-insect cell system
摘  要：
The objectives of this study were to express the native structure of fusion protein(F)and hemagglutinin glycoprotein(H)of canine distemper virus(CDV).The Fand H gene of lesser panda attenuated strain were amplified by PCR and cloned into pFastBacTM1 vector.The recombinant plasmids were sequenced,and then were transformed into competent DH10 BacTM E.coli cells to construct shuttle plasmids,rBacmid F and rBacmid H,by homologous recombination.Therecombinant plasmids were then transfected into Sf9 cells to construct recombinant baculoviruses and the expression products of rF and rH were identified with IFA and Western blot.The expression of rF and rH in insect cells infected with recombinant baculoviruses were identified by IFA with dog anti-CDV hyperimmune serum and were detected by Western blot with mouse polyclonal antibody against F and H major epitopes of CDV.The molecular weight of expressed F and H protein were identified as 63 and 68kD,which were consistent with the expected value.The results show that both envelope glycoproteins are successfully expressed in baculovirus-insect cell expression system and have a good immunoreactivity.Our study laid the foundation for the development of the CDV virus-like particle vaccine.

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