鹅副粘病毒HN及F基因在昆虫杆状病毒表达系统的表达
摘要
鹅副粘病毒(Goose Paramyxovirus，GPMV)与新城疫病毒(New castle disease virus，NDV)基因核苷酸同源性来看，GPMV与NDV属于同种病毒，GPMV是NDV的一个变异，而不是一个新种病毒，是NDV毒株在水禽上引起发病，且病毒毒力加强。但是，GPMV与传统的NDV又有不同，差异主要在血凝素-神经氨酸酶蛋白(HN)及融合蛋白(Fusion protein，F)上，常规的NDV疫苗不能提供有效的保护，因此从分子生物学水平上对该病进行研究具有重要的意义。HN和F蛋白是GPMV诱导体液免疫的重要靶抗原，在抗病毒免疫中起着重要作用，无疑HN和F基因已成为研制GPMV基因工程苗及免疫监测试剂的首选基因。
本试验根据GenBank所发表的HN及F基因的cDNA序列，利用Primer5.0软件，参照昆虫细胞杆状病毒表达系统的质粒图谱，设计引物，以本实验室所保存的pMD18-T-HN及pMD18-T-F阳性质粒为模板扩增出HN及F基因，分别命名为pMD18-T-GPMV-HN及pMD18-T-GPMV-F。将其克隆至昆虫杆状病毒转移载体pBlueBacHis2A的XhoⅠ和EcoRⅠ之间的多克隆位点上，分别命名为pBlue-GPMV-HN及pBlue-GPMV-F。纯化该重组质粒并与杆状病毒共转染Sf9昆虫细胞，4d后获得重组病毒，名为P1。将经过3轮蚀斑筛选纯化，PCR鉴定获得重组病毒，分别命名为rBv-HN及rBv-F。将重组病毒反复扩毒3次，获得高浓度的重组病毒名为P3，接种Sf9单层细胞上，4d后收获病变细胞，经SDS-PAGE电泳鉴定，表达F蛋白分子量约为60ku，表达HN蛋白分子量约为66ku，与理论值相符。表达产物HN蛋白的Western blot和Dot-ELISA结果表明其可与鸡抗GPMV血清发生特异性抗原反应，证实所表达的HN蛋白有较好的反应原性。
本研究将株的HN及F基因利用昆虫杆状病毒表达系统中表达，并获得了HN主要抗原位点基因的表达，对GPMV的基础性研究、预防控制和病毒诊断试剂研制奠定重要的物质基础。
Goose Paramyxovirus(GPMV)is homologous with New castle disease virus(NDV) at a greatsignificance, GPMV was revealed to be a variant of NDV but not a new virus. One strain of NDVinfect waterfowl and the virulence was enhanced. However, GPMV was not absolutely differentwith NDV, hemagglutinin-neuraminidase(HN) and fusion protein(F)protein were mutant and resultin the invalidation of traditional vaccine. Therefore, the study on the virus at molecular degree wasof great significance. HN and F proteins were the primary target antigens of humoral immuneresponse against virus. HN and F proteins have become the preferred genes for production ofgenetically engineering vaccine and pathogen diagnosis.
In this research, one set of primers was designed based on the sequence of HN and F gene ofGPMV of GenBank and the sequence of eukaryotic transfer vector pBlueBachis2A using primer 5.0. HN and F gene was amplified from pMD18-T-HN and pMD18-T-F respectively and designatedas pMD18-T-GPMV-HN and pMD18-T-GPMV-F. They were ligated into the transfer vectorpBlueBachis2A with XhoⅠand EcoRⅠrestriction sites, and the recombinant plasmids weredesignated as pBlue-GPMV-HN and pBlue-GPMV-F. Co-transfection of Sf9 insect cell wasperformed using purified recombinant plasmids and Bacmid-N-blue DNA. Recombiant virusnamed P1 was recovered 4d after transfection. After 3 serial passges of plaque assay screenfollowed by PCR confirmation, the recombinant virus were designated as rBv-HN and rBv-F. After3 passages of propagation of the recombinant virus, high-titer virus named P3 was used to inoculateSf9 cells followed by harvest after 4d. The F and HN proteins were confirmed to be 60ku and 66kuin length which were consistent with the speulated size as judged by SDS-PAGE seperation. HNprotein could be specifically recognized by chicken-anti-GPMV antibodies as investigated byWestern blot and Dot-ELISA assays. All the results proved the better reactionogenicity of HNprotein.
In this research, HN and F proteins of GPMV/JS/1/97/Go strain were expressed withbaculovirus expression system. The expression of the antigenic sites of HN protein wasconfirmed. The results of this study provided data for fundamental research on GPMV, preventivecontrol and virus diagnosis.
本试验根据GenBank所发表的HN及F基因的cDNA序列，利用Primer5.0软件，参照昆虫细胞杆状病毒表达系统的质粒图谱，设计引物，以本实验室所保存的pMD18-T-HN及pMD18-T-F阳性质粒为模板扩增出HN及F基因，分别命名为pMD18-T-GPMV-HN及pMD18-T-GPMV-F。将其克隆至昆虫杆状病毒转移载体pBlueBacHis2A的XhoⅠ和EcoRⅠ之间的多克隆位点上，分别命名为pBlue-GPMV-HN及pBlue-GPMV-F。纯化该重组质粒并与杆状病毒共转染Sf9昆虫细胞，4d后获得重组病毒，名为P1。将经过3轮蚀斑筛选纯化，PCR鉴定获得重组病毒，分别命名为rBv-HN及rBv-F。将重组病毒反复扩毒3次，获得高浓度的重组病毒名为P3，接种Sf9单层细胞上，4d后收获病变细胞，经SDS-PAGE电泳鉴定，表达F蛋白分子量约为60ku，表达HN蛋白分子量约为66ku，与理论值相符。表达产物HN蛋白的Western blot和Dot-ELISA结果表明其可与鸡抗GPMV血清发生特异性抗原反应，证实所表达的HN蛋白有较好的反应原性。
本研究将株的HN及F基因利用昆虫杆状病毒表达系统中表达，并获得了HN主要抗原位点基因的表达，对GPMV的基础性研究、预防控制和病毒诊断试剂研制奠定重要的物质基础。
Goose Paramyxovirus(GPMV)is homologous with New castle disease virus(NDV) at a greatsignificance, GPMV was revealed to be a variant of NDV but not a new virus. One strain of NDVinfect waterfowl and the virulence was enhanced. However, GPMV was not absolutely differentwith NDV, hemagglutinin-neuraminidase(HN) and fusion protein(F)protein were mutant and resultin the invalidation of traditional vaccine. Therefore, the study on the virus at molecular degree wasof great significance. HN and F proteins were the primary target antigens of humoral immuneresponse against virus. HN and F proteins have become the preferred genes for production ofgenetically engineering vaccine and pathogen diagnosis.
In this research, one set of primers was designed based on the sequence of HN and F gene ofGPMV of GenBank and the sequence of eukaryotic transfer vector pBlueBachis2A using primer 5.0. HN and F gene was amplified from pMD18-T-HN and pMD18-T-F respectively and designatedas pMD18-T-GPMV-HN and pMD18-T-GPMV-F. They were ligated into the transfer vectorpBlueBachis2A with XhoⅠand EcoRⅠrestriction sites, and the recombinant plasmids weredesignated as pBlue-GPMV-HN and pBlue-GPMV-F. Co-transfection of Sf9 insect cell wasperformed using purified recombinant plasmids and Bacmid-N-blue DNA. Recombiant virusnamed P1 was recovered 4d after transfection. After 3 serial passges of plaque assay screenfollowed by PCR confirmation, the recombinant virus were designated as rBv-HN and rBv-F. After3 passages of propagation of the recombinant virus, high-titer virus named P3 was used to inoculateSf9 cells followed by harvest after 4d. The F and HN proteins were confirmed to be 60ku and 66kuin length which were consistent with the speulated size as judged by SDS-PAGE seperation. HNprotein could be specifically recognized by chicken-anti-GPMV antibodies as investigated byWestern blot and Dot-ELISA assays. All the results proved the better reactionogenicity of HNprotein.
In this research, HN and F proteins of GPMV/JS/1/97/Go strain were expressed withbaculovirus expression system. The expression of the antigenic sites of HN protein wasconfirmed. The results of this study provided data for fundamental research on GPMV, preventivecontrol and virus diagnosis.
引文
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李文良，卞汝霖，冯太兰等．1999．鹅类新城疫病原研究[J]．畜牧与兽医．31(2)：1-3
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曹殿军，苑纯秀，郭鑫等．1999．新城疫病毒F48E9株融合蛋白基因核苷酸序列分析[J]．中国预防兽医学报．21(2)：103-106．
陈金顶，辛朝安，任涛等．2000．鹅源禽副粘病毒GPMV／QY97-1株的生物学特性[J]．中国兽医学报．20(2)：128-130．
丁壮．2001．新城疫病毒HN蛋白基因的克隆、序列分析、表达及其免疫原性研究[D]中国人民解放军军需大学．
高宏丽．2004．鹅副粘病毒HN和F基因克隆、序列分析及原核表达载体的构建[D]．东北农业大学．
李文良，卞汝霖，冯太兰等．1999．鹅类新城疫病原研究[J]．畜牧与兽医．31(2)：1-3
李玉峰，王永坤，严维巍等．2000．禽副粘病毒Ⅰ型结构蛋白的分析[J]．山东家禽．5：7-9．
廖延雄．1991．兽医微生物实验诊断手册[M]．北京：中国农业出版社．741-744．
刘光磊，王宝维．2003．我国鹅副粘病毒病的研究进展[J]．山东家禽．4：39-42．
刘华雷，王永坤，严维巍等．2002．鹅副粘病毒F蛋白基因的克隆及序列分析[J]．中国预防兽医学报．22(6)：404-407．
刘华雷，王永坤，周继宏等。2000．鹅副粘病毒毒力特性的研究[J]．中国预防兽医学报．22(3)：164-169．
刘禄，韦平，韦天超等．2003．禽Ⅰ型副粘病毒病快速诊断及广西分离株F基因研究初报[J1．广西畜牧兽医．19(2)：59-61．
钱忠明，周继宏，朱国强等．1999．鹅副粘病毒病流行病学和血清学研究[J]．中国家禽．21(10)：6-8．
任涛，陈金顶．1998．鹅的禽副粘病毒(GPMV)感染的研究[A]．中国畜牧兽医学会禽病学分会第九次学术研讨会论文及[C]．广西．107-110．
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