表达小反刍兽疫H蛋白的重组山羊痘病毒疫苗  

Recombinant goat pox virus expressing PPRV H protein

作　　者：陈伟业[1] 曲林茂[2] 胡森[1] 胡倩倩[2] 张倩[1] 支海兵[3] 黄克和[2] 步志高[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室农业部兽医公共卫生重点开放实验室,哈尔滨150001 [2]南京农业大学动物医学院,南京210095 [3]中国兽医药品监察所,北京100081

出　　处：《生物工程学报》2009年第4期496-502,共7页Chinese Journal of Biotechnology

基　　金：国家高技术研究发展计划(863计划)(No.20060102Z449)资助~~

摘　　要：本研究旨在构建表达小反刍兽疫H蛋白的重组山羊痘病毒,并评价其免疫效力。利用筛选基因gpt和eGFP并结合空斑技术,纯化筛选了重组小反刍兽疫H基因的重组山羊痘病毒(rGPV-PPRV-H),经过PCR鉴定重组病毒已纯化。免疫荧光和蛋白印迹都表明重组病毒能够感染绵羊羔羊睾丸细胞表达小反刍兽疫H蛋白。以2×106PFU的rGPV-PPRV-H皮内注射免疫山羊6只,并于首次免疫后28d以相同剂量进行二次免疫。免疫后采血分离血清进行病毒中和试验,结果表明,一次免疫后21d,山羊痘病毒中和抗体效价依次为40、80、≥80、≥80、40、≥80,和小反刍兽疫病毒中和抗体效价全部转阳依次为80、80、80、80、40、40、10;二次免疫后14d,山羊痘病毒中和抗体效价抗体全部大于或等于80,小反刍兽疫病毒中和抗体效价依次为≥80、80、≥80、80、80、40,应该具有对山羊痘病毒和小反刍兽疫病毒强毒攻击的完全免疫保护作用。本研究为小反刍兽疫重组山羊痘疫苗的产业化提供了参考。The purpose of the study is to construct recombinant goat pox virus （GPV） expressing Peste des petits ruminants virus （PPRV） H protein, and to evaluate the immunization effect. Recombinant GPV containing PPRV H gene （rGPV-PPRV-H） was selected and purified by gpt and eGFP utilizing plaque purification, and the final selected recombinant GPV was proved to be purified by PCR. Immunofluorescence and Western blotting showed that the recombinant virus could express H protein of PPRV while infecting lamb testis cells. Six goats were immunized with 2×10^6pFU rGPV-PPRV-H through intradermal injection, and were immunized for the second time at 28 days with the same dose recombinant virus after first immunization. Serum was collected after immunization, and was analyzed for the neutralization antibodies. 21 days after first immunization, the neutralization antibodies of GPV were 40, 80,≥80, ≥80, 40, ≥80 in turn, and neutralization antibodies of PPRV were 80, 80, 80, 80, 40, 40, 10 in turn; 14 days after second immunization, the neutralization antibodies of GPV were all ≥80, and the neutralization antibodies of PPRV were 〉80, 80, 〉80, 80, 80 and 40 in turn. This study established a foundation for the industrialization of the PPRV recombinant GPV vaccine.

关 键 词：重组疫苗 小反刍兽疫 山羊痘病毒载体 中和抗体 

分 类 号：S852.5[农业科学—基础兽医学] Q789[农业科学—兽医学]