【作者】 印春生；

【导师】 夏春；

【作者基本信息】 中国农业大学 ， 预防兽医学， 2014， 博士

【摘要】 口蹄疫(Foot and mouth disease, FMD)和小反刍兽疫(Peste des petits ruminants, PPR)是严重危害山羊、绵羊等家养和野生反刍动物的两种烈性病毒性传染病,分别由口蹄疫病毒(FMDV)和小反刍兽疫病毒(PPRV)引起。在我国,FMD和PPR均被列为一类动物传染病,疫苗免疫是防控此两种疫病的重要手段。灭活疫苗广泛应用于羊的FMD免疫预防,但其存在免疫期短、成本昂贵、不能完全阻止病毒在体内复制等不足。PPR弱毒疫苗N75/1株已被证明安全、有效,广泛应用于亚、非等PPR流行国家和地区。包括中国在内的广大发展中国家同时存在FMD和PPR流行,给养羊业造成重大经济损失。采用N75/1株为载体,研制表达FMDV保护性抗原的重组病毒活疫苗,有可能实现一种活疫苗预防PPR和FMD两种疫病,不仅有利于降低FMD防疫成本,提高FMD免疫覆盖率,而且弥补了灭活疫苗缺乏激活细胞介导免疫反应的不足。本研究旨在验证上述疫苗技术策略的可行性。为此,本研究首先以亚洲1型FMD疫苗毒株所提取的RNA为模板,以合成的VP1特异性引物进行RT-PCR扩增,获得了该毒株的VP1基因,并将其连接至pET-21a载体后进行表达。将纯化的VP1蛋白免疫大耳白兔制备了多克隆抗体,ELISA测定其抗体效价为1：8192。同时,应用表达纯化的VP1蛋白作为包被抗原建立了检测牛亚洲1型FMDV抗体的间接ELISA方法。再利用已经建立的PPRV/N75/1株反向遗传操作系统,构建了表达亚洲1型FMDV-VP1蛋白的重组PPRV。构建简要过程为先在pN75/1-insertion质粒载体的P和M基因之间插入了亚洲1型FMDV-VP1基因,构建了pN75/1-VP1重组质粒,再将pN75/1-VP1质粒、pCA-N、 pCA-P和pCA-L共转染入Vero细胞,获得了重组病毒,命名为rPPRV-VP1。rPPRV-VP1在Vero细胞上的生长曲线与PPRV/N75/1相似,病毒含量达107.7TCID50/mL; IFA和Western-blot分析证实VP1蛋白在rPPRV-VP1感染Vero细胞获得正确表达；将rPPRV-VP1在Vero细胞上连续传25代次,对不同代次病毒液的病毒含量测定和RT-PCR分析,证明rPPRV-VP1在传代过程中能够保持稳定表达重组VP1蛋白,显示出良好的遗传稳定性。为验证rPPRV-VP1对免疫靶动物的安全性和研究抗体持续期,采用6×107TCID50剂量颈部皮下接种5只山羊,持续观察28日。结果表明rPPRV-VP1对山羊具有良好的安全性；同时以6×106TCID50接种山羊后定期采血检测PPRV和FMDV中和抗体,结果表明,接种后第56日两种病毒中和抗体效价均达到峰值,并能够维持到接种后140日无明显下降。最后对rPPRV-VP1的免疫原性及其对FMDV的攻毒保护效力进行了评价。rPPRV-VP1和N75/1疫苗株分别按6×106TCID50剂量,分别颈部皮下接种健康易感山羊和牛,接种后定期采血检测PPRV和FMDV的中和抗体,并在免疫后接种亚洲1型FMDV强毒进行FMD攻毒保护效力测定。羊的试验结果为,接种后第14日,所有rPPRV-VP1接种羊PPRV和亚洲1型FMDV中和抗体均转为阳性；PPRV/N75/1接种照羊PPRV中和抗体全部转阳,但FMDV中和抗体保持阴性；两组羊的PPRV中和抗体滴度无显著差异。接种后第40日时进行攻毒,观察10日,接种PPRV/N75/1的4只羊均表现出典型FMD症状；接种rPPRV-VP1的6只羊中,4只羊观察期间无任何口蹄疫症状,另2只羊的发病时间延迟,且症状较PPRV/N75/1组羊明显较轻。牛的试验结果为,接种rPPRV-VP1的5头牛均诱导产生显著的亚洲1型FMDV中和抗体,而PPRV/N75/1接种的2头牛均未产生FMDV抗体。接种后第21日时进行攻毒,观察10日,接种PPRV/N75/1的2头牛均表现典型的FMD症状,接种rPPRV-VP1的5头牛中有2头未发现FMD症状,另3头牛和PPRV/N75/1接种牛相比FMD发病时间推迟,症状显著减轻。总结,本研究利用PPRV/N75/1株反向遗传系统,构建了表达亚洲1型FMDV-VP1的重组病毒rPPRV-VP1。rPPRV-VP1接种山羊安全性良好；接种山羊和牛均可诱导产生PPRV和FMDV中和抗体,并能够产生对亚洲1型FMDV强毒的攻击的免疫保护。结果证明,PPRV/N75/1株作为构建FMD活载体疫苗具有技术可行性,有可能实现一种活病毒疫苗同时预防PPR和FMD两种重大动物疫病。更多还原

【Abstract】 Foot-and-mouth disease (FMD) and Peste-des-petits ruminants (PPR) are both severe and highly contagious diseases of domestic (goats and sheep) and wild ruminants, which caused by foot-and-mouth disease virus (FMDV) and peste-des-petits ruminants virus (PPRV) respectively, and both classified into list I of animal diseases in China. There are no effective medical therapies to treat the PPR and FMD, but only through vaccines inoculation to prevent and control. Inactivated vaccine is used widely to control FMD in goats and sheep, however, it is expensive and has a short immunity duration. PPR attenuated vaccine (N75/1strain) has been confirmed to be safe and effective potency to prevent the PPR disease in prevalent countries of Africa and Asia. In developing countries, including China, both FMD and PPR are epidemic and cause great losses every year. A recombinant live vaccine expressing protective antigen of FMDV with PPRV (N75/1strain) vector might prevent PPR and FMD at the same time. The combined vaccine could not only reduce prevention costs and increase immune coverage, but also make up the shortfall of inactivated vaccine to activate the cell-mediated immune response. This study would be aim to testify feasibility of the above strategy.For this object, total RNA was firstly extracted from the strain of FMD (type Asial) inactivated vaccine and the first strand cDNA were synthesized by reverse transcription. The VPl gene was obtained from the cDNA by PCR and cloned into the pET-21a plasmid for expressing in E. coli BL21. The purified recombinant VP1protein was inoculated rabbits to prepare polyclonal antibody for several times. The titer of the prepared antibody was1:8192in indirect ELISA. Furthermore, indirect ELISA was established using the expressed VP1protein as coating antigen to detect antibody against FMDV (type Asial) in bovine sera.Secondly, based on the established reverse genetics systems of PPRV (N75/1), the recombinant PPRV expressing VPl gene of FMDV (type Asial)was constructed. In brief, VP1gene was inserted between P and M gene of PPRV/N75/1clone, then inserted into pN75/1-insertion and named pN75/1-VP1. The pN75/1-VP1was transfected into Vero cells together with the helper plasmids of pCA-N, pCA-P and pCA-L is using lipofatamine2000. The recombinant virus was rescued and named rPPRV-VP1. The growth curves of rPPRV-VP1were similar to PPRV (N75/1) in Vero cells, up to107.7TCID50/mL. Analysis of IFA and Western-blot showed that the recombinant virus expressed antigens of PPRV and VP1of FMDV. During the25successive passages in Vero cells, the recombinant virus kept stably expression of VP1. In inoculated goats with rPPRV-VP1, the antibody against PPRV and FMDV were both detected, titers were up to peak in56dps, and kept stably to140dps. In inoculated goats with an overdose of rPPRV-VP1, all the goats were healthy in the28days, which indicated that rPPRV-VP1was safe for goats.Goats and bovines were inoculated subcutaneously with6×106TCID50doses of rPPRV-VP1, respectively. In the goat experiments, the sera were collected on different days after vaccination and VNA titers against PPRV and FMDV were determined. The results showed that antibody against PPRV and FMDV were both conversed to positive after14days inoculation in the rPPRV-VP1vaccine group. The VNA titers against PPRV among the four time points of the two groups were not significantly different. However, the VNA titers against FMDV among the four time points were very significantly different At the40th day, all of the two group goats were inoculated virulent FMDV strain to evaluate the challenge potency. In the rPPRV-VP1vaccines group, four of the six goats had no clinical symptoms of FMD in the end of observation periods and the other two had a less serious symptoms than the four PPRV/N75/1vaccines control goats. In the bovine experiments, the sera were also collected on different days after vaccination and VNA titers against PPRV and FMDV were tested, and at the21st day, all of the bovines were inoculated virulent FMDV strain. The results showed that the VNA titers against PPRV among the three time points of the two groups were either not significantly different, however, the VNA titers against FMDV were also very significantly different. In the challenge test, two of the five bovines(2/5) had no clinical symptoms of FMD in the end of observation periods and the other three had less serious symptoms than the two PPRV/N75/1vaccnined control bovines.In summary, the study constructed a recombinant rPPRV-VP1to express VP1protein of FMDV (type Asial) with the established reverse genetics systems of PPRV(N75/1). The recombinant rPPRV-VPl had a good safety and could stimulate vaccined goats or bovines to produce antibodies against PPRV and FMDV at the meantime, furthermore, it could protect them entirely or partly from a virulent FMDV challenge. The studies showed that the novel FMDV recombinant vector vaccine using PPRV(N75/1strain) was feasible and might be an attractive candidate vaccine for preventing PPR and FMD simultaneously.更多还原