【作者】 苏春霞；

【导师】 陈溥言；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2007， 博士

【摘要】 口蹄疫（foot-and-mouth disease，FMD）是由口蹄疫病毒（foot-and-mouth diseasevirus，FMDV）引起的烈性传染病，主要感染偶蹄目动物。除动物死亡造成直接经济损失外，动物在患病期间肉和奶的生产停止导致该地区甚至该国家的畜产品进出口贸易停止，也造成巨大的经济损失。因此，世界各国都很重视本病的防制。FMD弱毒疫苗和灭活疫苗等常规疫苗都具有良好的免疫原性，在预防和控制FMD的过程中发挥着重要作用，但由于病毒毒力返强、病毒灭活不彻底、活病毒逃逸疫苗加工厂等不安全因素。促使人们寻求一种更加安全有效的FMD疫苗。近年来，随着分子生物学技术的飞速发展，FMD基因工程疫苗等多种新型的疫苗都在不断的研究开发中，并取得了很好的成绩，但存在的问题是价格昂贵和动物保护试验不尽如人意。因此，研究者试图从多方面增强FMD疫苗的免疫效果，其中用细胞因子作为分子免疫佐剂，已引起众多研究者的关注。研究表明，许多细胞因子具有明显的免疫佐剂效应，可增强机体对抗原的反应性或增强抗原的免疫原性。细胞因子在体内缓慢而持久地释放，不会对机体产生毒性作用。具有佐剂效应的细胞因子有干扰素（IFN-α、IFN-β、IFN-γ）；淋巴因子（IL-2、IL-3、IL-4、IL-5、IL-10等）；单核因子（IL-1、IL-6、IL-8、IL-12等）及其它细胞因子。本研究的目的是通过酵母表达系统和非复制型腺病毒载体系统表达口蹄疫病毒抗原基因及共表达抗原基因和免疫佐剂，研究免疫佐剂对重组蛋白和重组病毒的免疫增强作用，为口蹄疫基因工程疫苗的研究探索新的途径。研究主要从以下几个方面展开。1．人工合成O型口蹄疫病毒VP1-2A基因，并克隆到pMDl8-T载体中，命名为pMD18-VP1-2A（上海Invitrogen公司完成），以pMD18-VP1-2A为模板，设计一对引物，PCR扩增VP1基因，亚克隆到pET-32a（+）中，构建了表达载体pET-VP1，通过IPTG诱导表达，实现了融合蛋白VP1在大肠杆菌中的高效表达，表达融合蛋白为44kD左右，Western-blot试验表明，该重组蛋白具有免疫反应性。以纯化的重组VP1蛋白作为包被抗原，抗原最佳包被浓度确定为10.56μg／ml，能明显的区分口蹄疫病毒的阴阳性血清，建立了检测FMDV抗体的ELISA方法，为后期的研究打下基础。2．人工合成O型口蹄疫病毒的多表位基因，并克隆到pMD18-T载体中，命名为pMD18-EG（上海Invitrogen公司完成）。另外，以含有人结核杆菌HSP70基因的质粒（pMD18-HSP70）为模板，设计引物PCR扩增HSP70基因。将目的基因克隆到酵母表达载体pPICZaA中，先后构建四个酵母表达载体pPICZaA-EG、pPICZaA-VP1、pPICZaA-EG-HSP70、pPICZaA-VP1-HSP70。通过电穿孔法将四个表达载体转化到酵母菌X-33中，用Zeocin⁺平板筛选重组子，甲醇诱导表达，经SDS-PAGE电泳鉴定四个重组蛋白的大小分别约为23kD（rVP1），20kD（rEG），89kD（rEG-HSP70），92kD（rVP1-HSP70），免疫印迹分析后表明重组蛋白均具有免疫反应性。将四个重组蛋白以皮下接种的方式给小鼠进行3次免疫，同时设两组对照组小鼠，分别免疫PBS和常规灭活疫苗，然后通过测定IgG、IgGl、IgG2a、淋巴细胞增殖功能和IL-4、IFN-γ水平来衡量重组蛋白的免疫效果。结果表明重组蛋白既能诱导细胞免疫应答又能诱导体液免疫应答，rEG-HSP70产生的抗体与常规灭活苗相当，而细胞免疫水平从高到低依次为rEG-HSP70，rVP1-HSP70，灭活疫苗，rEG，rVP1。提示，HSP70作为分子佐剂在口蹄疫基因工程疫苗的研究中具有潜在的应用前景，为口蹄疫基因工程疫苗的研究提供一种新的思路。3．重新设计引物扩增VPl和多表位基因，在多表位基因中加入通用型辅助性T淋巴细胞表位（PADRE），并命名为EGS，分别将VP1和EGS基因克隆到腺病毒的穿梭载体pAdenoVator-CMV5-IRES-GFP中。得到的阳性穿梭质粒经PmeⅠ酶线性化后，利用电转化技术将线性化的质粒与腺病毒骨架载体pAdeno Vator△E1／E3共转化大肠杆菌BJ5183感受态细胞，使其在大肠杆菌中进行同源重组。将筛选到的重组病毒质：粒经PacⅠ酶线性化后暴露出包装信号，通过脂质体Lipofectamine ^TM2000转染293A细胞后得到重组病毒。利用报告基因GFP检测病毒滴度和感染效率，重组病毒rAd5-EGS和rAd5-VP1的滴度分别为10^10.62个TCID₅₀／ml和10^11.87个TCID₅₀／ml，重组病毒经过连续五代传代后效价稳定。通过PCR和western-blot证实FMDV V1l及表位基因得到成功的表达并有很好的免疫反应性。4．分别以质粒pMD18-VP1-2A和以含有猪白细胞介素-2基因（PoIL-2）的质粒pMD18-PoIL-2为模板，PCR扩增VP1-2A和poIL-2基因，分别亚克隆到腺病毒的穿梭载体pAdenoVator-CMV5-IRES-GFP中。分别得到的阳性穿梭质粒pAd5polL-2和pAd5VP1-2A-PolL-2，经PmeⅠ酶线性化后，利用电转化技术将线性化的质粒与腺病毒骨架载体pAdenoVator△E1／E3共转化大肠杆菌BJ5183感受态细胞，使其在大肠杆菌中：进行同源重组。将筛选到的重组病毒质粒经PacⅠ酶线性化后暴露出包装信号，通过脂质体Lipofectamine ^TM2000转染293A细胞后得到重组病毒。Western-blot试验证实在FMDV 2A蛋白的介导下VP1-2A-PoIL-2裂解为VP1-2A和PoIL-2。同时，用淋巴细胞增殖试验分析单独表达PoIL-2和串联表达VP1-2A-poIL-2均具有刺激淋巴细胞增殖的生物活性。5．以含有猪IFN-a基因的质粒pMD18-poIFN-a为模板，PCR扩增出poIFN-a基因，克隆到含有VP1-2A基因的腺病毒的穿梭载体pAdeno Vator-CMV5-IRES-GFP中。得到的阳性穿梭质粒pAd5VP1-2A-POIFN-a，经PmeⅠ酶线性化后，利用电穿孔法将其与腺病毒骨架载体pAdenoVator△E1／E3共转化大肠杆菌BJ5183感受态细胞，使其在大肠杆菌中进行同源重组。将筛选到的重组病毒质粒经PacⅠ酶线性化后暴露出包装信号，通过脂质体Lipofectamine ^TM2000转染293A细胞后得到重组病毒。Western-blot证实VP1-2A-PoIFN-a得到了表达，并在FMDV 2A蛋白的介导下VP1-2A-PoIFN-a裂解为VP1-2A和POIFN-a。细胞病变抑制法测定结果表明，重组猪a-干扰素具有较高抗病毒活性，10⁵U／ml。6．6周龄小鼠随机分为14组，每组12只。将6个重组病毒rAd5VP1、rAd5EGS、rAd5poIL-2、rAd5poIFN-a、rAd5VP1-2A-poIFN-a，rAd5VP1-2A-poIL-2分别以腹膜内接种的方式给小鼠进行3次免疫，同时设两组对照组小鼠，分别免疫PBS和常规灭活疫苗。然后通过测定IgG、IgG1、IgG2a、淋巴细胞增殖功能和IL-4、IFN-γ水平来衡量重组病毒的免疫效果。结果表明重组病毒既能诱导细胞免疫应答又能诱导体液免疫应答，rAd5VP1-2A-poIFN-a，rAd5VP1-2A-poIL-2免疫组的效果更优于其它免疫组，证实细胞因子发挥了明显的免疫佐剂效应。也进一步证实我们选用口蹄疫病毒2A蛋白介导细胞因子表达是正确的，因为只有2A发挥了有效的裂解作用，才能避免细胞因子在其氨基端融合其他蛋白而使构象发生改变，构象的改变可能会使它的功能和活性受到影响。抗体亚型测定结果显示，重组病毒既能诱导IgG1的分泌又能诱导IgG2a的分泌，但以IgG1为主。同时细胞因子ELISA试验结果进一步表明重组病毒主要刺激Th2分化（IL-4），刺激Th1（γ-IFN）的分化的能力相对弱些。以上实验证明，选择口蹄疫病毒2A蛋白酶介导口蹄疫病毒抗原基因与免疫佐剂在非复制型腺病毒载体表达系统中进行串联表达的设计思路是合理的，为口蹄疫基因工程疫苗的研究提供新的思路和见解。更多还原

【Abstract】 Foot-and-mouth disease （FMD） was caused by FMDV, is a highly contagious, acute vesicular disease of cloven-hoofed animals, and is responsible for large economic losses, both as a direct result of the disease, and because of the necessary restrictions imposed on animal products and trade for disease control. Many researchers in the world pay attention to prevent and control this disease.Although both of attenuated FMD vaccine and inactivated vaccine are effective, major problems were encountered such as unpredictable virulence in the field, high-containment vaccine production facilities, carried a slight risk of residual contamination of inactivated vaccines with live virus. Its use would most probably complicate the discrimination of naturally infected and vaccinated animals. Several novel approaches to vaccine development are emerging, and these may enjoy significant advantages over conventional vaccines. However, as yet they remain too costly for general veterinary use, and their immunogenicity is often limited. One way to remove this shortcoming is to improve the adjuvants used in the formulation of the vaccine. In general, there are some potent adjuvants, such as interferon（IFN-a、IFN-β、IFN-γ）; lymph cytokines（IL-2、IL-3、IL-4、IL-5、IL-10）; monocytines（IL-1、IL-6、IL-8、IL-12） and other cytokines.In this study, the antigen of FMDV and some adjuvants （HSP70, PoIL-2, PoIFN-a） were co-expressed in methylotrophic yeast expression system and adenovirus vector expression system respectively. The affection of adjuvants on FMDV recombinant gene-engineering vaccine in mice was detected. This will provide novel ways for study of foot-and-mouth newly vaccine. All studies were listed as below:1. VP1-2A gene of FMDV type O was synthesized and cloned into pMD18-T, named pMD18-VP1-2A （It had been done by Shanghai Invitrogen company） . VP1 gene was amplified from pMD18-VP1-2A and subcloned to pET-32a（+）, named pET-VP1, recombinant VP1 was expressed in E.coli induced with IPTG. Recombinant protein was testified by SDS-PAGE, and which was expressed successfully and relative molecular mass was about 44kD. Western-blot result indicated that the recombinant protein had specific immunogenic characteristic against antibody of FDMV. ELISA test indicated that 10.56μg/ml of purified recombinant VP1 protein was coated as antigen can distinguish positive serum and negative serum of FMDV. This study was the basic for further study.2. Multi-epitopes genes of 0 type foot-and-mouth diseases virus was synthesized and cloned into pMD18-T and named pMD18-EG （It had been done by Shanghai Invitrogen company）. M.tuberculosis HSP70 gene was amplified from pMD18-HSP70. VP1 gene, EG gene and HSP70 gene were cloned into yeast expression vector pPICZaA. Four recombinant vectors were constructed and named pPICZaA-EG、pPICZaA-VP1、pPICZaA-EG-HSP70、pPICZaA-VP1-HSP70 respectively. Recombinant vector were transformed into methylotrophic yeast Pichia pastoris X-33 by electrophoration. The recombinant transformants were selected by Zeocin and induced by the addition of methanol every 24 h. The result of SDS-PAGE indicated that the recombinant proteins were successfully expressed in methylotrophic yeast Pichia pastoris and relative molecular mass were about 23kD（rVP1）, 20kD（rEG）, 89kD（rEG-HSP70）, 92kD（rVP1-HSP70）. Western blotting indicated that four recombinant proteins had specific antigenicity of FDMV. Mice were inoculated transcutaneous three times at a two-week interval with fusion protein, PBS and conventional inactivated vaccines. Level of IgG、IgG1、IgG2a、IL-4、IFN-γand proliferation T lymphocyte test were detected to evaluate humoral immune and cellular immune responses. The results indicated that fusion protein could elicit specific humoral immune and cellular immune responses. Compared with conventional inactivated vaccines, fusion protein elicited close to FMDV antibody level. Cellular immune responses in turn is rEG-HSP70, rVP1-HSP70, inactivated vaccine, rEG, rVP1, which suggest that HSP70 as molecular adjuvant can improve the immunogenicity of FMDV recombinant vaccine, thus provide valuable support for further development of FMDV genetic engineering vaccines.3. VP1 gene and multi-epitopes gene were synthesized and cloned into pAdeno Vator-CMV5-IRES-GFP, named pA5-EGS and pA5-VP1 respectively. pA5-EGS and pA5-VP1 were Linearized by Pme I, and then linearized vector and pAdeno VatorΔE1/E3 co-transformed into E.coli BJ5183 competent cells by electrophoration. The homologous recombinant plasmids pAd5-EGS and pAd5-VP1 were selected and Linearized by Pac I to expose the encapsidation signal. Linearized virus plasmids were transfected into 293A cells by Lipofectamine ^TM2000, and recombinant viruses were named rAd5-EGS and rAd5-VP1. Infection titer and rate were evaluated through monitoring green fluorescent protein （GFP） expression. The filter of rAd5-EGS and rAd5-VP1 are 10^10.62个TCID₅₀/ml and 10^11.87个TCID₅₀/ml respectively. The recombinant viruses were passaged 5 times in 293A and the titer is still stable.. Expression of VP1 and multi-epitopes of FMDV were testified by PCR and western-blot, wetern-blot result indicated that expression products had specific antigenicity of FDMV.4. VP1-2A gene of FMDV was and porcine Interleukin-2 gene was amplified from pMD18-VP1-2A and pMD18-poIL-2（pMD18-T contains porcine interleukin-2） respectively and cloned into pAdeno Vator-CMV5-IRES-GFP, named pA5VP1-2A-PoIL-2 and pA5-PoIL-2. Recombinant vectors were linearized by Pme I, and then linearized recombinant vectors and pAdeno VatorAEl/E3 co-transformed into E.coli BJ5183 competent cells by electrophoration. The homologous recombinant plasmids were selected and linearized by Pac I to expose the encapsidation signal. Linearized virus plasmids were transfected into 293A cells by Lipofectamine?2000, and recombinant viruses were named rAd5-VP1-2A-PoIL-2 and rAd5-PoIL-2. Western-blot indicated that rAd5-VP1-2A-PoIL-2 had specific antigenicity of FDMV and VP1-2A-PoIL-2 was cleaved by 2A with VP1-2A and PoIL-2. The lymphocyte proliferation test indicated that rPoIL-2 had biological activity.5. Porcine IFN-a （PoIFN-a） gene was amplified from pMD18-poIFN-a, and cloned into pAdenoVator-CMV5-IRES-GFP vector containing VP1-2A gene, named pAd5VP1-2A-PoIFN-a, which was linearized by Pme I, and then linearized pAd5VP1-2A-PoIFN-a and pAdeno VatorAEl/E3 co-transformed into E.coli BJ5183 competent cells by electrophoration. The homologous recombinant plasmids pAd5VP1-2A- PoIFN-a was selected and Linearized by Pac I to expose the encapsidation signal. Linearized virus plasmids were transfected into 293A cells by Lipofectamine?2000, and recombinant viruses were named rAd5VP1-2A-PoIFN-a. Western-blot indicated that rAd5-VP1-2A-poIFN-a had specific antigenicity of FDMV and VP1-2A-PoIFN-a was cleaved by 2A with VP1-2A and PoIFN-a.. Recombinant virus was verified to be of high PoIFN-a activity by inhibiting the cyto-pathogenic effect, 10⁵U/ml. 6. 6 weeks old female BALB/c mice were randomly separated into 14 groups, 12 mice per group, mice were inoculated intraperitoneal with recombinant viruses（rAd5VP1、rAd5EGS、rAd5poIL-2、rAd5poIFN-a、rAd5VP1-2A-poIFN-a and rAd5VP1-2A-poIL-2） three times at 2 weeks intervals, and with PBS and conventional inactivated vaccines as control. Level of IgG、IgG1、IgG2a、IL-4、IFN-γand proliferation T lymphocyte test were detected to evaluate humoral immune and cellular immune responses of recombinant viruses. The results indicated that rAd5VP1-2A-poIFN-a, rAd5VP1-2A-poIL-2 induced more stronger responses than inactivated vaccine done. The results indicated that cytokines （IFN-a and IL-2） enhanced immunity of recombinant virus. FMDV 2A separated the polyprotein into its single proteins and thus provide a maximal degree of freedom for cytokines activity, recombinant viruses showed VP1-2A-PoIL-2 and VP1-2A-PoIFN-a at a size of 25 kDa in Western blot analyses using an anti-FDMV antibody, revealing a proper cleavage of the fusion protein. ELISA test of antibody sub-type results indicated that recombinant viruses induced secretion both of IgG1 and IgG2a, partial to IgG1.The seem result elicited from cytokines ELISA test, secretion of both of Th1（γ-IFN） and Th2（IL-4） were induced by recombinant viruses, partial to Th2. This study demonstrates the VP1-2A-PoIL-2 （VP1-2A-PoIFN-a） fusion construct to be an efficient tool encoding two functions in one open reading frame. This promising candidate can be for instance in viral vectors vaccine studies. Thus provide valuable support for further development of FMD genetic engineering vaccines.更多还原