【作者】 贾晓娟；

【导师】 杨汉春； 刘文军；

【作者基本信息】 中国农业大学 ， 预防兽医， 2014， 博士

【摘要】 Gp96是热休克蛋白HSP90家族一员,具有多种免疫学效应,包括提高抗原呈递、刺激抗原呈递细胞(APCs)产生细胞因子、激发机体特异性免疫应答及细胞毒性T细胞(CTL)反应,以及免疫佐剂效应。本论文主要研究猪Gp96N对猪繁殖与呼吸综合征病毒(PRRSV)的B、T细胞表位合成肽疫苗、B细胞表位串联亚单位疫苗以及猪圆环病毒Ⅱ型(PCV2)灭活疫苗的免疫佐剂效应。首先建立了表达猪Gp96N的大肠杆菌和毕赤酵母p.pastoris-X33表达系统,利用纯化的猪Gp96N作为免疫佐剂,在小鼠和猪上,从细胞免疫、体液免疫和天然免疫角度,分析了猪Gp96N对上述三种疫苗的免疫增强效应。研究结果为猪Gp96N作为免疫佐剂在疫苗中的应用提供了科学依据。从猪肾脏中提取RNA,采用RT-PCR扩增得到猪Gp96N基因cDNA片段,分别插入载体pET-32a和pPICZαA中,构建出原核表达载体pET-32a/Gp96N和真核表达载体pPICZaA/Gp96N,分别转入大肠杆菌BL21和毕赤酵母p.pastoris-X33中,经抗性筛选获得重组大肠杆菌BL21/pET-32a/Gp96N和重组毕赤酵母p.pastoris-X33/pPICZaA/Gp96N。对表达产物进行纯化,获得猪Gp96N蛋白。根据文献报道,筛选出PRRSV的结构蛋白和非结构蛋白的高度保守B细胞和T细胞抗原表位,经化学合成表位多肽,与猪Gp96N联合免疫小鼠和猪。结果发现,猪Gp96N能够增强表位肽诱导的特异性体液免疫和细胞免疫应答；猪Gp96N能显著上调IL-12和TNF-a,下调IL-4和IL-10的表达水平；用PRRSV高致病性毒株JXwn06对免疫猪的攻毒试验结果表明,表位多肽+猪Gp96N (BT-Gp组)免疫猪在攻毒后第7天全部死亡,表位多肽免疫(BT组)猪在第5天全部死亡,而对照组(PBS组和猪Gp96N组)的猪在攻毒后第3天全部死亡。虽然BT-Gp组和BT组的免疫猪均不能抵抗JXwn06的攻击,但比较攻毒后猪体温、临床症状、血清中病毒滴度等发现,在PRRSV攻毒后7天内,猪Gp96N与表位多肽联合使用能在一定程度上缓解猪的临床症状,降低猪血清中的病毒载量。将一些PRRSV的结构蛋白和非结构蛋白Nsp2的高度保守B细胞抗原表位通过柔性linker连接,利用重叠PCR技术获得融合基因,并将其重组到原核表达质粒中,经过大量表达和纯化后得到串连多B细胞表位亚单位疫苗(Cpl、Cp2)。以猪Gp96N为免疫佐剂,与Cpl、CP2联合免疫小鼠和猪。经过三次免疫后,采用ELISA检测小鼠和猪血清中的抗体。结果表明,免疫小鼠和猪血清中均可检测到PRRSV特异性抗体,且猪Gp96N能提高抗体滴度3-4倍；从免疫小鼠血清中可检测到中和抗体,但在猪血清中未检测到中和抗体；ELISPOT及淋巴细胞增殖试验结果表明,猪Gp96N能显著提高疫苗诱导的细胞免疫。此外,定量ELISA检测发现,猪Gp96N能显著上调猪血清中IFN-γ, IL-12等Th1型细胞因子以及TNF-α的表达水平,同时能够下调IL-4和IL-10的表达水平。用不同剂量的猪Gp96N与PCV2灭活疫苗联合免疫猪,采用ELISA检测免疫前后各组血清中PCV2特异性抗体IgG以及IFN-γ、TNF-α、IL-1β、IL-6等细胞因子含量。结果发现,与单独免疫PCV2灭活苗及商业化的亚单位疫苗相比,猪Gp96N能够提高特异性抗体IgG水平,且随着Gp96N剂量增加,IgG水平呈递增趋势,表明猪Gp96N能够增强PCV2灭活疫苗的体液免疫。此外,与单独免疫疫苗相比,猪Gp96N+PCV2灭活疫苗联合免疫,能提高免疫猪的IFN-γ、TNF-α水平,但对1L-1β、IL-6没有影响。综上研究结果表明,猪Gp96N在一定程度上能够增强PRRSV亚单位疫苗的体液和细胞免疫应答,提升PCV2灭活疫苗的体液免疫效果,具有潜在的免疫佐剂效应。更多还原

【Abstract】 The Gp96, a member of heat shock protein (HSP) family, displays various immunological functions includingstimulating the expression of cytokines by activating the antigen presentation cells in innate immunity, and eliciting an antigen-specific cytotoxic T lymphocyte (CTL) immune response to eliminate pathogens and tumors by facilitating antigen cross-presentation in adaptive immunity, as well as acting as immunological adhuvant. In this study, we focused on the immunological effects of Gp96N on synthetic peptides and subunit vaccinesof porcine reproductive and respiratory syndrome virus (PRRSV) and inactivated vaccine of porcine circovirus type2(PCV2). Porcine Gp96N was first expressed by two expression systems of E.coliBL21/pET-32a/Gp96N and p.pastoris-X33/pPICZaA/Gp96N.Then purified Gp96was used as adjuvant in combination with the three vaccines in mice and piglets in order toanalyze the immunoenhancement effect of porcine Gp96N. Our results provide theoretical basis for the application and development of Gp96in vaccines.RNA was extracted from porcine kidney to amplify the cDNA ofGp96gene-encoded N-terminal22-370amino acids (aa) with RT-PCR.The amplified product was cloned into the expression vector pET-32a and pPICZaA, and then the recombinant plasmids were transformed into E.coli/BL21and p.pastoris-X33, respeCtively.The transformants were screened on resistance plates and named E.coliBL21/pET-32a/Gp96N and p.pastoris-X33/pPICZaA/Gp96N.The expressed protein (termed Gp96N) was purified.Two B-cell epitopes and seven T-cell epitopes on nonstructural and structural proteins of PRRSV and a Pan DR T-helper cell epitope were synthesized and mixed with the Gp96N as an adjuvant, and then immune responses were evaluated in mice and piglets. The results showed that Gp96N could enhance the humoral and cellular immune effects of PRRSV synthetic peptides. Moreover, it could significantly up-regulate the expression levels of IL-12and TNF-α and down-regulate the levels of IL-4and IL-10. Following challenge with the virulent PRRSV isolate JXwn06, the piglets vaccinated with the mixture of Gp96N and synthenic peptides presented milder clinical symptoms, lower viremia, and less pathological lesions in their lungs compared with the piglets immuned with the peptides alone although all the vaccinated piglets in each group died, suggesting the Gp96N can alleviate the clinical symptoms of challenging piglets in some extent.Two multi-epitope subunit vaccines, named as Cpl and Cp2, were designed based on the conserved B cell epitopes of PRRSV proteins.The Gp96N was used as the adjuvant. Immune responses elicited by the different combinations of Cpl/Cp2and Gp96N were examined in mice and piglets. The results indicated that the group of Cpl/Cp2-Gp96N (CG) combination could induce3-4-fold higher titers of Cpl/Cp2-ELISA antibodies and neutralizing antibodies (NAs) in mice than the groups received Cpl/Cp2immunization alone or with Freund’s adjuvant. Additionally, Gp96N significantly enhanced the levels of lymphocyte proliferative responses of splenocytes or peripheral blood mononuclear cells from the vaccinated mice or piglets. The production of IFN-y in mice splenocytes, TNF-a, IFN-y, and IL-12in sera of piglets were also remarkably increased with the treatment of Gp96N, while IL-4was reduced by half and IL-10was decreased to an undetectable level. These results suggest that the porcine Gp96N can effectively enhance the innate and adaptive immune responses of Cpl/Cp2with a Thl-type bias. Therefore, the multi-epitope subunit vaccine Cpl/Cp2co-administered with porcine Gp96N might potentially be a promising candidate vaccine for the prevention and control of PRRSV in pigs.Mice and piglets were immuned with different doses of Gp96N combined with inactivated vaccine of PCV2. The titer of PC V2-specific-IgG and the expression level of some cytokines in serum including IFN-y,TNF-a,IL-1β and IL-6were tested by ELISA kit. The results showed that Gp96N could significantly increase titers of IgG antibody compared to the groups immunized with the inactivated and commercial vaccines of PCV2alone. In addition, Gp96N could significantly elevate the levels of IFN-y and TNF-a, but had no effects on the levels of IL-1β and IL-6in sera of piglets.Taken together, all the findings indicate that Gp96N can enhance humoral and celluar responses of epitope peptide and subunit vaccines of PRRSV, and improve humoral immunological effect on inactivated vaccine of PCV2in some extent, suggesting that Gp96shares the potential immune effect for vaccines as an adjuvant.更多还原