【作者】 邵军军；

【导师】 柳纪省； 常惠芸； 吴润；

【作者基本信息】 甘肃农业大学 ， 预防兽医学， 2011， 博士

【摘要】 口蹄疫是由口蹄疫病毒引起偶蹄兽猪、牛、羊等主要经济畜种发病的重大动物疫病。FMDV灭活疫苗作为FMD综合防控体系的重要组成部分,为有效防控和净化口蹄疫做出了十分重要的贡献,但灭活疫苗也存在诸多的缺点和不足,如需要区分疫苗免疫动物和自然感染动物,需要建设生物安全实验室;最为严重的是,病毒有逃逸生产车间的危险。分子生物学技术的发展以及合成肽免疫原性的证实,为研制FMDV抗原表位疫苗提供了理论依据和技术支持,人们对FMDV抗原表位疫苗进行了广泛的研究,在小动物模型显示出了良好的免疫效力,但是对本动物的免疫效果并不理想。近年来我国口蹄疫疫情有所抬头,出现A,O,Asia 1多个血清型交替流行的态势,严重地威胁我国畜牧业的健康发展,为了有效控制和净化在我国流行的FMDV,本研究针对我国目前流行的Asia 1型和O型FMDV毒株,利用分子生物学,蛋白质工程及其相关技术进行羊口蹄疫病毒Asia 1多表位疫苗和猪口蹄疫病毒O型多表位疫苗的研究,为我国有效防控FMD提供优质安全的疫苗储备。针对目前流行的Asia 1型江苏毒株,设计并合成了病毒主要抗原VP1的135-160肽段和197-211肽段的重复串联表位基因(RE)。采用RT-PCR技术克隆了羊的免疫球蛋白重链恒定区基因(OIgGC)和Asia 1病毒的3D基因,将3D、RE、OIgGC分别或融合插入pET-30a构建了一系列重组表达质粒pRE-OIgGC,p3D,pOIgGC,将RE插入pGEX-4T-1构建了pGST-RE重组表达质粒,并实现了这些重组蛋白在E.coli的规模化表达及高通量纯化,所有重组蛋白以包涵体形式表达。Western blotting结果显示,重组蛋白RE-OIgGC,3D和GST-RE能与FMDV阳性血清发生免疫反应,OIgGC能与兔抗羊血清发生免疫反应。将适量的重组抗原与等体积的ISA206佐剂混合制备一系列疫苗,即OIgGC(100μg/ml),RE-OIgGC(100μg/ml),RE-OIgGC(100μg/ml)+3D(50μg/ml)和GST-RE(100μg/ml)。通过免疫豚鼠和羊体,测定血清中和抗体效价、淋巴细胞增殖实验和攻毒保护实验对候选疫苗进行免疫效力评价。免疫豚鼠结果显示,除了PBS和OIgGC组外,重组蛋白RE-OIgGC,RE-OIgGC+3D和GST-RE均能诱导产生保护性中和抗体,加强免疫后能保护动物抵抗103 ID50口蹄疫病毒的攻击。添加FMD病毒3D的多表位疫苗组的中和抗体水平最高,与灭活疫苗诱导的中和抗体效价没有明显的不同。以GST和为载体蛋白的重组抗原免疫豚鼠后血清中和抗体并没有明显差异。另外,添加3D蛋白的疫苗组诱导了最高水平的淋巴细胞增殖,充分说明了3D蛋白具有改善体液免疫反应和诱发T细胞免疫反应的能力。羊免疫结果显示,以宿主自身分子IgG重链恒定区为载体蛋白的2组多表位疫苗的中和抗体水平明显高于以GST分子为载体蛋白组,加强免疫后血清中和抗体效价明显快速升高,说明OIgGC发挥了生物学功能,明显改善了多表位抗原的免疫原性,而3D蛋白的免疫佐剂功能不是特别明显。设计并合成了FMD病毒O型主要抗原VP1的140-160肽段和200-213肽段的重复串联表位基因(RE)。利用RT-PCR扩增了猪的免疫球蛋白重链恒定区基因(SIgGC)和O型病毒的3D基因,将3D、RE、SIgGC分别或融合插入pET-22b成功构建了重组表达质粒pRE-SIgGC和p3D,并实现了RE-SIgGC和3D重组蛋白在E.coli的规模化表达及高通量纯化,以包涵体形式表达的重组蛋白经Western blotting证实具有免疫反应性。RE-SIgGC和3D重组蛋白经纯化后按照适当比例进行混合,并与与氢氧化铝、完全弗氏佐剂和ISA206等多种佐剂配伍成10种不同形式的猪口蹄疫病毒O型多表位疫苗,通过本动物猪免疫效力试验筛获了一种候选疫苗,单剂免疫猪体后免疫效力达到甚至优于传统灭活疫苗。随后用试制的三批疫苗进行了候选疫苗的最小免疫剂量、免疫效力(PD50)、保存期和免疫持续期以及抗体消长情况测定,全面评价疫苗的免疫效力。结果显示,用抗原含量为83μg的疫苗免疫猪,不仅能诱发机体产生高滴度的保护性中和抗体,而且能保护猪只抵抗103 ID50口蹄疫病毒感染,5/5保护;三批候选疫苗的PD50为5.20~10.05达到甚至高于国家标准(3.0 PD50);免疫动物后30天,血清中和抗体达到峰值,此后,随时间的延长中和抗体效价开始下降,免疫后7个月内血清中和抗体水平仍然维持在较高水平,大多数(70%)免疫动物能够抵抗病毒的感染;疫苗于2~8℃保存12个月并不影响疫苗的免疫效力。这些结果充分说明了本研究研制的猪口蹄疫病毒O型多表位疫苗是一种具有良好开发前景的疫苗,有望替代灭活疫苗用于我国口蹄疫病毒的防控。总之,通过本研究我们成功设计、构建了Asia 1和O型口蹄疫病毒的多表位疫苗的组装体系和表达体系,并实现了重组抗原的规模化原核表达和高通量纯化。通过引入具有重要免疫学功能的宿主自身分子IgG和具有佐剂功能的FMDV的3D蛋白,明显提高了多表位疫苗的免疫效力,并诱发了T细胞免疫反应。通过豚鼠和本动物羊和猪筛获了具有良好免疫效力的候选疫苗,尤其是筛获的猪口蹄疫病毒O型多表位候选疫苗不仅具有良好免疫效力,而且抗原用量少、免疫持续期长,疫苗在2~8℃保存稳定,是一种十分具有开发前景的疫苗,对其他动物病毒抗原表位疫苗的研制具有借鉴意义。更多还原

【Abstract】 Foot-and-mouth (FMD) is a highly contagious and economically devastating viral disease of cloven-hoofed live-stock including pig, cattle, sheep and goats that causing by foot-and-mouth disease virus (FMDV). The inactivated FMDV vaccines as one of the systematic implements of prevention and control disease play a crucial role for control and eradication of FMD. However, the inactivated vaccines have several disadvantages including difficulty to discriminate between infected and vaccinated animals, requiring of the safe building biologically. Seriously, there is a potential risk of live virus escaping from vaccine plants. Development of the molecular biology and validation of immunogenicity of the synthetic peptides offer the theories and techniques for development of antigenic epitope vaccines to FMDV. To date, there were a lots of reports about antigenic epitope vaccines to FMDV and their immune potential is promising in the small model. However, they confer the limited immune responses in the natural hosts. In recent years, outbreaks of FMD frequently occur in China, particularly, several FMDV serotypes including types Asia 1, O and A alternately occurred, which the severely impact to the live-stock and animal’s products in China. With a aim to control and eradicate FMDV occurred in China, the multiple-epitope vaccines to FMDV type Asia 1 in sheep and FMDV type O in swine have been developed with the molecular biology, proteins engineering and relative techniques, based on the current prevalent FMDV isolates type O and Asia 1. These vaccines as a safe and efficacy alternative will redound to prevention and control of FMD in China in the future.We designed and synthesized a tandem-repeated multiple-epitopes gene (RE) based on residues 135-160 and 197-211 of FMDV VP1 type Asia 1 Jiangsu isolate occurred in China. The ovine immunoglubine G heavy constant region gene (OIgGC) and 3D of FMDV type Asia 1 were amplified by RT-PCR. A series of the recombinant expression plasmids, pRE-OIgGC, p3D and pOIgGC, were constructed by directly inserting RE-OIgGC, 3D and OIgGC into an expression vector, pET-30a, respectively. The RE was purified and subcloned into an expression vector, pGEX-4T-1, which resulted in a recombinant plasmid, pGST-RE. These recombinant proteins were large-scale expressed in E.coli and the high-throughput purification proteins were obtained. All of the recombinant proteins were expressed as a formation of inclusion bodies in E.coli. Western blotting results showed that the recombinant RE-OIgGC, 3D and GST-RE are able to react with anti-FMDV positive serum, and the OIgGC is able to recognized by rabit anti-goat IgG. A series of vaccines, OIgGC(100μg/ml),RE-OIgGC(100μg/ml),RE-OIgGC(100μg/ml)+3D(50μg/ml) and GST-RE(100μg/ml)were performed by emulsification of appropriate proteins with a equal volume of ISA206. Potency of immunity of vaccine candidates in guinea pigs and sheep was evaluated by detection of neutralizing antibodies in serum, lymphocytes proliferation assay and virus challenge test. The results of the vaccinated guinea pigs showed that the recombinant proteins, RE-OIgGC, RE-OIgGC+3D and GST-RE, except groups PBS and OIgGC are able to elicit protective levels of neutralizing antibodies in the vaccinated guinea pigs and to offer compelete protection of guinea pigs against 103 ID50 of virus challenge. The multiple-epitope vaccine containing 3D is able to induce the highest titers of neutralizing antibodies than that of other proteins, which is the same high as that of the inactivated vaccine. Regretingly, the GST-RE elicit the same high levels of neutralizing antibodies as that of the RE-OIgGC. The possible explanation for this phenomenon is that the carrier proteins, GST and OIgGC, are the foreign proteins to the guinea pigs. In addition, the vaccine containing 3D is also able to the highest levels of lymphocytes proliferation than that of other vaccine candidates. These results demonstrated that 3D protein is not only able to improve the humoral immune responses, but to elicit T cell immune responses. The results of the vaccinated sheep showed that two groups, RE-OIgGC and RE-OIgGC+3D, elicit the significant higher level of neutralizing antibodies than that of GST-RE. The neutralizing antibodies is signifcantly rapid raise after booster vaccination compared with the initiation vaccination, which demonstrated that OIgGC play an important function biologically and significantly enhance the immunogenicity of the multiple-epitope vaccine. However, the immune adjuvant function of 3D protein was not especially distinctness in the vaccinated sheep. A tandem-repeated multiple-epitope gene (RE) containing residues 140-160 and 197-211 of VP1 of FMDV type O was designed and synthesized. A swine immunoglubine G heavy constant region (SIgGC) and 3D of FMDV type O were amplified by RT-PCR. The gene 3D, RE and SIgGC were subcloned into a expression vector, pET-22b, which resulted in the recombinant expression plasmids, pRE-SIgGC and p3D. The recombinant proteins, RE-SIgGC and 3D, were large-scale expressed in E.coli and highly throughput purified. The recombinant proteins were expressed as a formation of inclusion bodies in E.coli, and their immunoreactivity was verified with Western blotting. The RE-SIgGC and 3D was purified with Ni-NTA resin and a mixture of RE-SIgGC and 3D was developed according to an appropriate concentration ratio of two proteins. Subsequently, ten different multiple-epitopes vaccines to FMDV type O in swine were developed by emulsification of the mixture of proteins with a equal volume of several adjuvants including Al(OH)3, compelete Freund’s adjuvant and ISA206 etc. Potency of immunity of ten vaccines were evaluated by vaccinated swine, and then a vaccine candidate has been screened. The results showed that the potency of immunity in swine vaccinated with single-dose vaccination is the same or higher than that of the traditionally inactivated vaccine. Three sets of vaccine candidates have been developed in our laboratory and their potency of immunity were systematically evaluated including detection of the minimum immune dosage, potency of immunity (PD50), duration of storage, duration of immunity and variation of neutralizing antobides after vaccination. The results showed that a vaccine candidate containing 83μg mixture of antigen is able to elicite the higher titers of neutralizing antibodies and to offer compelete protection of swine agaisnt 103 ID50 of virus challenge, protective ratio is 5/5. Potency of immunity from three sets of vaccine candidates are about 5.20-10.05 PD50 that is higher than the country standard (3.0 PD50). The titers of neutralizing antibodies reach the peak at days 30 post-vaccination, and the titers of neutralizing antibodies are gradually decline over times. Inspiringly, at months 7 post-vaccination, the higher titers of neutralizing antibodies were remained and majority of the vaccinated swine (70%) were compelete protection against virus challenge. Potency of immunity is not affected when vaccine candidate stored at 2-8℃for 12 months. These results indicated that we have developed multiple-epitope vaccine to FMDV type O in swine is a promising vaccine candidate, which may be to replace the traditionally inactivated and to be use for the control and prevention of FMD in China in the future.Conclusions, we have successfully designed and constructed the systems of assembly and expression for the multiple-epitopes vaccines to FMDV type O and Asia 1. The recombinant proteins were large-scale expressed in E.coli and highly throughput purified proteins were obtained. An immunological important molecular, host self immunoglubine G, and 3D of FMDV as a adjuvant were considerated in the designing of multiple-epitopes vaccine, which significantly improve the potency of immunity of vaccine and elicite cell immune responses. The higher potency vaccine candidates were selected by vaccination of guinea pigs, sheep and pigs. Particularly, the selected multiple-epitope vaccine candidate to FMDV type O in swine has several advantages including a higher potency of immunity, lower amount of antigen, longer duration of immunity and good stability stored at 2-8℃. Therefore, we have developed vaccine candidate is a promising vaccine, which will be used for the reference to develep multiple-epitope vaccines to other animal’s viruses.更多还原