表达小反刍兽疫病毒H、F蛋白的重组山羊痘病毒的构建及免疫原性研究
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摘要
小反刍兽疫(Peste des petits of ruminants, PPR)是由小反刍兽疫病毒(Pestedes petits of ruminants virus, PPRV)引起的危害山羊、绵羊等小反刍动物的重大疫病,为世界动物卫生组织(The World Organisation for Animal Health)发布的A类烈性传染病之一。近年来,PPR在我国周边国家和地区及我国西藏自治区的肆虐对我国的养羊业构成严重的威胁。
目前,我国西藏和新疆等西部地区采用经Vero细胞传代弱化的PPRV弱毒疫苗来预防PPR。但是,弱毒疫苗免疫小反刍兽之后无法与野毒感染相区别,不利于对PPR的血清学监控,更重要的是,PPRV弱毒疫苗热敏感性强,限制了其在热带地区的应用。由于山羊痘(Goat pox, GP)与PPR宿主范围一致,地理分布区域类似,GPV弱毒疫苗具有较强的热稳定性,并且随着DNA重组技术的发展,山羊痘病毒(Goat pox virus, GPV)在做为疫苗载体方面显示出很强的优越性,国外已有将PPRV保护性抗原基因F／H插入山羊痘病毒疫苗载体构建二重疫苗进行免疫学研究的报道,并且显示了良好的应用前景,但在国内尚未见相关的报道。本研究将利用我国自行培育并得到广泛应用的GPV弱毒株,分别构建表达PPRV囊膜蛋白F和H的重组山羊痘病毒,并对其免疫原性进行了初步评估。
试验一、小反刍兽疫重组山羊痘病毒的构建。首先在已有载体pTK-gpt-gfp的基础上,构建了痘病毒晚期启动子p11驱动的、内部核糖体进入位点(ires)介导的黄嘌呤鸟嘌呤磷酸转移酶基因(gpt)和绿荧光蛋白基因(egfp)串联表达的转移载体pTK-gpt-ires-egfp,将PPRV保护性抗原H、F基因片段分别克隆至pTK-gpt-ires-egfp Sal I位点,获得载体质粒pTK-gig-PPRV-H、pTK-gig-PPRV-F;以我国自行培养并得到广泛应用的山羊痘病毒(GPV)疫苗株为亲本病毒,以山羊痘病毒(GPV)的复制非必需区TK基因为靶点,通过质粒pTK-gig-PPRV-H、pTK-gig-PPRV-F与GPV的同源重组,霉酚酸(Mycophenolic acid, MPA)药物筛选,病毒蚀斑单克隆化后获得完全纯化的重组病毒rGPV-PPRV-F和rGPV-PPRV-H。以F、H基因的特异性引物PCR鉴定,表明基因片段分别在重组病毒中正确整合；经Western-blot和IFA(间接免疫荧光)方法证实H蛋白、F蛋白分别在重组病毒中稳定表达。
试验二、rGPV-PPRV-F、rGPV-PPRV-H重组病毒的初步免疫研究。以2×106PFU的rGPV-PPRV-F、rGPV-PPRV-H皮内注射免疫山羊,一免后第28天进行加强免疫,采血分离血清进行病毒中和试验。结果表明,本研究构建的小反刍兽疫重组山羊痘病毒rGPV-PPRV-H、rGPV-PPRV-F一次免疫山羊,3周后PPRV中和抗体转阳率可达100%(6／6)、100%(3／3),二免后PPRV中和抗体效价进一步得到增强。参照OIE PPRV弱毒疫苗效力检验合格要求和国外相关文献的报道,本研究获得的重组病毒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,为小反刍兽疫重组山羊痘疫苗的产业化提供参考。
Peste des petits ruminants (PPR) is an acute and highly contagious disease of domestic and wild small ruminants caused by the Peste des petits ruminants virus (PPRV) in the genus Morbillivirus, family Paramyxoviridae. With high morbidity and mortality, it is one of the major notifiable diseases of the World Organisation for Animal Health. In recent years, some neighboring countries and regions of our country outbreak PPR, causing heavy economic losses and this constitutes a serious threat to small ruminants in our country.
Now, attenuated PPRV strain Nigeria/75/1 achieved through serial passages on Vero cells is used in our country to prevent PPR.However,the attenuated vaccine is not being able to differentiate non-infected but vaccinated animals from those which have been exposed to PPRV,and the more important is that attenuated PPR vaccine is thermolabile, the virus can retain thermostability when lyophilized with suitable stabilizers. With the advent of DNA recombinant technology, efforts are being made to develop effective PPR marker vaccines to enable such differentiation. Some people in the foreign countries constructed PPR poxvirus-based recombinant vaccines which can protest animals from capripox and PPR at the same time after immunizations. But, there was none relative reports in our country. In the present study, Vaccine strain of Goat pox virus (GPV) cultivated and widely used in domestic animals was employed as the Parents virus, and by homologous recombination between the tk gene of Goat pox virus and plasmid, we constructed recombinant goat pox virus expressing F/H protein of PPRV. At the same time, the immunogenicity of recombinant Goat pox virus were evaluated in this report.
Experiment 1. Construction of recombinant Goat pox virus expressing Peste des petits ruminants H/F protein.In this study, firstly the gene transfer vector pTK-gpt-ires-eGFP was constructed on the basic of vector pTK-gpt-GFP. In the former vector, there were two back to back promoters:p11 late promoter and p7.5 early/late promoter, which were flanked by the left and right arms of tk. In the expression cassette, there were one reporter gene(green fluorescent protein,eGFP) and one selective gene (xanthine-guanine phosphoribosyl transferase,gpt), and they both were controlled by the p11 late promoter. The anther promoter, p7.5 early/late promoter, controlled the expression of target protein. Secondly, the H and F gene from PPRV was cloned into the transfer vector pTK-gpt-ires-eGFP, respectively. By homologous recombination with live strain of GPV and pressure selection by adding Mycophenolic acid(MPA) in the culture, we obtained the recombinant GPV name as rGPV-PPRV-H、rGPV-PPRV-H,respectively.Finally, the inserts of F and H gene into the GPV genomes and their accuracy and stability were confirmed by PCR.The expression of F and H protein of PPRV was confirmed by immunofluorescence test and Western-blot.The results show that he recombinant goat pox virus expressing PPRV H/F protein were constructed and purified.
Experiment 2. The immunogenicity evaluation of recombinant Goat pox virus. Nine goats were immunized twice with 2×106 PFU rGPV-PPRV-H(six goats)、rGPV-PPRV-F(three goats) through intradermal injection, and Serum was collected after immunization, and was analyzed for the neutralization antibodies. The results show that 21 days after first immunization, the neutralization antibodies of GPV and PPRV were all positive; after second immunization, the neutralization antibodies of GPV and PPRV were all enhanced.
In conclusion, this study established the foundation for the development of the recombinant virus against PPRV and goat pox virus at the same time.
目前,我国西藏和新疆等西部地区采用经Vero细胞传代弱化的PPRV弱毒疫苗来预防PPR。但是,弱毒疫苗免疫小反刍兽之后无法与野毒感染相区别,不利于对PPR的血清学监控,更重要的是,PPRV弱毒疫苗热敏感性强,限制了其在热带地区的应用。由于山羊痘(Goat pox, GP)与PPR宿主范围一致,地理分布区域类似,GPV弱毒疫苗具有较强的热稳定性,并且随着DNA重组技术的发展,山羊痘病毒(Goat pox virus, GPV)在做为疫苗载体方面显示出很强的优越性,国外已有将PPRV保护性抗原基因F／H插入山羊痘病毒疫苗载体构建二重疫苗进行免疫学研究的报道,并且显示了良好的应用前景,但在国内尚未见相关的报道。本研究将利用我国自行培育并得到广泛应用的GPV弱毒株,分别构建表达PPRV囊膜蛋白F和H的重组山羊痘病毒,并对其免疫原性进行了初步评估。
试验一、小反刍兽疫重组山羊痘病毒的构建。首先在已有载体pTK-gpt-gfp的基础上,构建了痘病毒晚期启动子p11驱动的、内部核糖体进入位点(ires)介导的黄嘌呤鸟嘌呤磷酸转移酶基因(gpt)和绿荧光蛋白基因(egfp)串联表达的转移载体pTK-gpt-ires-egfp,将PPRV保护性抗原H、F基因片段分别克隆至pTK-gpt-ires-egfp Sal I位点,获得载体质粒pTK-gig-PPRV-H、pTK-gig-PPRV-F;以我国自行培养并得到广泛应用的山羊痘病毒(GPV)疫苗株为亲本病毒,以山羊痘病毒(GPV)的复制非必需区TK基因为靶点,通过质粒pTK-gig-PPRV-H、pTK-gig-PPRV-F与GPV的同源重组,霉酚酸(Mycophenolic acid, MPA)药物筛选,病毒蚀斑单克隆化后获得完全纯化的重组病毒rGPV-PPRV-F和rGPV-PPRV-H。以F、H基因的特异性引物PCR鉴定,表明基因片段分别在重组病毒中正确整合；经Western-blot和IFA(间接免疫荧光)方法证实H蛋白、F蛋白分别在重组病毒中稳定表达。
试验二、rGPV-PPRV-F、rGPV-PPRV-H重组病毒的初步免疫研究。以2×106PFU的rGPV-PPRV-F、rGPV-PPRV-H皮内注射免疫山羊,一免后第28天进行加强免疫,采血分离血清进行病毒中和试验。结果表明,本研究构建的小反刍兽疫重组山羊痘病毒rGPV-PPRV-H、rGPV-PPRV-F一次免疫山羊,3周后PPRV中和抗体转阳率可达100%(6／6)、100%(3／3),二免后PPRV中和抗体效价进一步得到增强。参照OIE PPRV弱毒疫苗效力检验合格要求和国外相关文献的报道,本研究获得的重组病毒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,为小反刍兽疫重组山羊痘疫苗的产业化提供参考。
Peste des petits ruminants (PPR) is an acute and highly contagious disease of domestic and wild small ruminants caused by the Peste des petits ruminants virus (PPRV) in the genus Morbillivirus, family Paramyxoviridae. With high morbidity and mortality, it is one of the major notifiable diseases of the World Organisation for Animal Health. In recent years, some neighboring countries and regions of our country outbreak PPR, causing heavy economic losses and this constitutes a serious threat to small ruminants in our country.
Now, attenuated PPRV strain Nigeria/75/1 achieved through serial passages on Vero cells is used in our country to prevent PPR.However,the attenuated vaccine is not being able to differentiate non-infected but vaccinated animals from those which have been exposed to PPRV,and the more important is that attenuated PPR vaccine is thermolabile, the virus can retain thermostability when lyophilized with suitable stabilizers. With the advent of DNA recombinant technology, efforts are being made to develop effective PPR marker vaccines to enable such differentiation. Some people in the foreign countries constructed PPR poxvirus-based recombinant vaccines which can protest animals from capripox and PPR at the same time after immunizations. But, there was none relative reports in our country. In the present study, Vaccine strain of Goat pox virus (GPV) cultivated and widely used in domestic animals was employed as the Parents virus, and by homologous recombination between the tk gene of Goat pox virus and plasmid, we constructed recombinant goat pox virus expressing F/H protein of PPRV. At the same time, the immunogenicity of recombinant Goat pox virus were evaluated in this report.
Experiment 1. Construction of recombinant Goat pox virus expressing Peste des petits ruminants H/F protein.In this study, firstly the gene transfer vector pTK-gpt-ires-eGFP was constructed on the basic of vector pTK-gpt-GFP. In the former vector, there were two back to back promoters:p11 late promoter and p7.5 early/late promoter, which were flanked by the left and right arms of tk. In the expression cassette, there were one reporter gene(green fluorescent protein,eGFP) and one selective gene (xanthine-guanine phosphoribosyl transferase,gpt), and they both were controlled by the p11 late promoter. The anther promoter, p7.5 early/late promoter, controlled the expression of target protein. Secondly, the H and F gene from PPRV was cloned into the transfer vector pTK-gpt-ires-eGFP, respectively. By homologous recombination with live strain of GPV and pressure selection by adding Mycophenolic acid(MPA) in the culture, we obtained the recombinant GPV name as rGPV-PPRV-H、rGPV-PPRV-H,respectively.Finally, the inserts of F and H gene into the GPV genomes and their accuracy and stability were confirmed by PCR.The expression of F and H protein of PPRV was confirmed by immunofluorescence test and Western-blot.The results show that he recombinant goat pox virus expressing PPRV H/F protein were constructed and purified.
Experiment 2. The immunogenicity evaluation of recombinant Goat pox virus. Nine goats were immunized twice with 2×106 PFU rGPV-PPRV-H(six goats)、rGPV-PPRV-F(three goats) through intradermal injection, and Serum was collected after immunization, and was analyzed for the neutralization antibodies. The results show that 21 days after first immunization, the neutralization antibodies of GPV and PPRV were all positive; after second immunization, the neutralization antibodies of GPV and PPRV were all enhanced.
In conclusion, this study established the foundation for the development of the recombinant virus against PPRV and goat pox virus at the same time.
引文
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