VP22短肽融合猪细小病毒样粒子的制备及免疫原性初步研究

发布时间：2018-07-08 12:09

本文选题：猪细小病毒 + VP2蛋白　；参考：《广西大学》2017年硕士论文

【摘要】：猪细小病毒(Porcine parvovirus,PPV)是引起母猪繁殖障碍的主要病原之一,疫苗免疫仍是预防控制该病的主要手段。病毒样粒子(Virus-like particles,VLPs)疫苗以其安全性高、免疫原性好成为各类病毒疫苗研究的热门方向。PPV病毒样粒子(PPV-VLPs)是不含PPVDNA的空衣壳结构,由PPVVP2(下文简称:VP2)结构蛋白体外自行组装形成,形态上与天然病毒粒子相似,具有很强的免疫原性和生物学活性。单纯疱疹病毒1型(HSV-1)VP22蛋白是该病毒的结构蛋白,不仅具有细胞间自由转运的能力,而且还可以促进融合抗原交叉致敏,增强免疫原性。此外,HSV-1VP22(下文简称:VP22)蛋白还可能提高DNA疫苗的免疫反应。本研究分为三个部分,详细研究内容如下:1、细胞穿膜肽VP22对生物膜穿透性的研究以载体pEGFP-N1为模板扩增增强型绿色荧光蛋白(EGFP)基因,通过SOE-PCR分别将VP22基因片段拼接到EGFP基因的3'/5'端,获得VP22-EGFP和EGFP-VP22融合基因;将融合基因及EGFP基因分别克隆到pET-28a(+)载体构建重组表达质粒 pET28a-EGFP-VP22、pET28a-VP22-EGFP和pET28a-EGFP,并进行测序分析及酶切鉴定,将鉴定正确的各重组表达质粒及pET-28a空载体分别转化宿主菌E.coli BL21(DE3)进行表达,表达产物经SDS-PAGE及Western Blot分析结果可见相对分子量分别为约31 kDa、31 kDa及27kDa的蛋白分子条带,大小与预期相符,表明EGFP-VP22、VP22-EGFP、EGFP蛋白表达成功。对表达的蛋白进行Ni-NTA亲和层析纯化,将纯化后的蛋白添加到Vero细胞中进行共培养,l0h后荧光显微镜下观察到加入VP22-EGFP和EGFP-VP22蛋白的细胞有绿色荧光,而加入EGFP的细胞与空白对照组细胞中则没有观察到绿色荧光存在,提示细胞穿膜肽VP22可有效携带与其融合的EGFP蛋白穿过细胞膜进入细胞,VP22蛋白融合到EGFP蛋白N端或C端并不影响VP22的穿膜效果。2、VP22短肽融合猪细小病毒样粒子的制备及鉴定利用Bac-to-Bac杆状病毒表达载体系统进行PPV VLPs的制备。以PPV N株的基因组为模板扩增其VP2基因,通过SOE-PCR分别VP22基因拼接到VP2基因片段的3'/5'端,获得VP22-VP2和VP2-VP22融合基因;将两种融合基因及VP2基因分别克隆到pFastBacTMl载体,获得重组质粒pFB-VP22-VP2、pFB-VP2-VP22 及 pFB-VP2。重组质粒转化至 DHlOBac 感受态细胞进行同源重组,获得重组杆粒Bacmid-VP22-VP2、Bacmid-VP2-VP22及Bacmid-VP2。重组杆粒经脂质体法转染sf9细胞,获得重组杆状病毒株rBac-VP22-VP2、rBac-VP2-VP22及rBac-VP2。随后将重组杆状病毒株感染到sf9细胞进行重组蛋白的表达,表达产物经SDS-PAGE和Western-blot鉴定分析,结果可见相对分子量分别为约67 kDa、67 kDa及64 kDa的蛋白分子条带,大小与预期相符,表明VP2-VP22、VP22-VP2及VP2蛋白表达成功。进一步用PPV单抗对重组杆状病毒感染细胞进行IFA鉴定,可观察到特异性荧光,再次证实重组蛋白的表达且具有生物活性。表达产物经透射电镜观察,VP2和VP22-VP2均可自我组装形成22-24nm、形态类似天然PPV的病毒样粒子;VP2-VP22没有观察到病毒样粒子,表明VP2的N端融合VP22不影响VP2形成病毒粒子的特性,而C端融合外源蛋白可能影响VP2形成病毒样粒子。3、VP22短肽融合猪细小病毒样粒子免疫原性的初步评价用饱和硫酸铵法初步纯化病毒样粒子VP22-VP2(VLPs-VP22-VP2)、病毒样粒子VP2(VLPs-VP2)及重组蛋白VP2-VP22,分别配以弗氏佐剂、ISA 206佐剂及白油司本佐剂制成疫苗免疫小鼠,并以PPV油乳灭活苗和PBS为对照,比较分析三种重组蛋白的免疫原性及筛选较好的免疫佐剂。通过间接ELISA检测免疫小鼠血清中PPV特异性抗体,结果显示VLPs-VP22-VP2及VLPs-VP2能有效诱导小鼠产生特异性PPV抗体,其配以ISA 206佐剂免疫产生的抗体水平与PPV灭活苗基本相当,而重组蛋白VP2-VP22产生的抗体水平要明显低于PPV灭活苗组;IL-2是T细胞增殖的主要生长因子,IFN-γ仅由活化T细胞和自然杀伤细胞(NK细胞)产生,检测这两个指标的不同程度能反映动物机体的细胞免疫水平,对采集的免疫小鼠血清IL-2和IFN-γ含量测定结果显示,与阴性对照组相比,VLPs-VP22-VP2、VLPs-VP2及重组蛋白VP2-VP22免疫组均能有效刺激IL-2和IFN-γ的产生,且融合VP22的VLPs-VP22-VP2刺激机体产生IFN-γ和IL-2的水平显著高于VLPs-VP2及PPV灭活苗(P0.05),此外,以ISA 206为佐剂,其免疫增强的效果要优于其他佐剂(P0.05)。上述结果表明,融合VP22蛋白可明显提高VP2蛋白的特异性T淋巴细胞的免疫应答水平,但却不能显著提高VP2蛋白的体液免疫效果;VP2N端融合VP22蛋白的免疫效果要优于其C端融合。VLPs-VP2、VLPs-VP22-VP2配以ISA 206佐剂免疫小鼠后能更有效地诱导机体产生针对PPV的体液及细胞免疫。
[Abstract]:Porcine parvovirus (PPV) is one of the main pathogens causing sow reproductive disorders. Vaccine immunization is still the main means to prevent and control the disease. Virus like particles (Virus-like particles, VLPs) vaccines have high safety and good immunogenicity as the hot direction of all kinds of disease vaccine,.PPV virus like particles (PPV-VLPs). The structure of empty capsid without PPVDNA is assembled by PPVVP2 (hereinafter referred to as VP2) structural protein in vitro. It is similar to natural virus particles, and has strong immunogenicity and biological activity. Herpes simplex virus 1 (HSV-1) VP22 protein is the structural protein of the virus, not only with the ability of free transport between cells, but also the ability of free transport between cells. It can promote the cross sensitization of the fusion antigen and enhance the immunogenicity. In addition, HSV-1VP22 (hereinafter referred to as VP22) protein may also improve the immune response of the DNA vaccine. This study is divided into three parts. The detailed research contents are as follows: 1, the cell penetrating peptide of membrane peptide VP22 amplified the enhanced green fluorescent egg with the carrier pEGFP-N1 as the template. The white (EGFP) gene was spliced into the 3'/5'end of the EGFP gene by SOE-PCR, and the fusion gene of VP22-EGFP and EGFP-VP22 was obtained. The fusion gene and EGFP gene were cloned into the pET-28a (+) vector to construct the recombinant expression plasmid pET28a-EGFP-VP22, pET28a-VP22-EGFP and pET28a-EGFP, and the sequencing analysis and enzyme digestion were carried out. The recombinant expression plasmids and pET-28a empty vectors were identified to express the host bacteria E.coli BL21 (DE3) respectively. The results of SDS-PAGE and Western Blot analysis showed that the molecular weight of the relative molecular weight was about 31 kDa, 31 kDa and 27kDa, the size of the protein molecule was in accordance with the predate, indicating that EGFP-VP22, VP22-EGFP, and protein were expressed. Ni-NTA affinity chromatography was used to purify the expressed protein, and the purified protein was added to Vero cells for co culture. After l0h, the cells with VP22-EGFP and EGFP-VP22 protein were observed to have green fluorescence, and the green fluorescence was not observed in the cells with EGFP and in the blank control group. The membrane peptide VP22 can effectively carry the EGFP protein which is fused with the cell membrane and enter the cell membrane. The fusion of VP22 protein to the N end or C end of the EGFP protein does not affect the membrane effect.2 of the VP22. The preparation and identification of the VP22 short peptide fusion porcine parvovirus like particles are prepared by using the Bac-to-Bac baculovirus expression vector system for PPV VLPs. The VP2 gene was amplified by the template, and the fusion gene of VP22-VP2 and VP2-VP22 was obtained by splicing the VP22 gene of SOE-PCR into the 3'/5'end of the VP2 gene fragment respectively. The two fusion genes and VP2 genes were cloned to the pFastBacTMl vector, and the recombinant plasmid pFB-VP22-VP2, pFB-VP2-VP22 and pFB-VP2. recombinant plasmid were transformed to the sense of sensation. The recombinant Bacmid-VP22-VP2, Bacmid-VP2-VP22 and Bacmid-VP2. recombinant rods were transfected into Sf9 cells by liposomes, and the recombinant baculovirus strain rBac-VP22-VP2, rBac-VP2-VP22 and rBac-VP2. subsequently infected the recombinant baculovirus strain to the Sf9 cells to express the recombinant protein, and the expression products were expressed in SDS-P. AGE and Western-blot identification analysis showed that the molecular weight of relative molecular weight was about 67 kDa, 67 kDa and 64 kDa protein bands were in accordance with expectations, indicating that the expression of VP2-VP22, VP22-VP2 and VP2 protein was successful. Further use PPV monoclonal antibody to identify the recombinant baculovirus infected cells and observe the specific fluorescence and reconfirm the weight of the recombinant baculovirus. The expression of histone and biological activity. The expression product can be self assembled to form 22-24nm, which is similar to the virus like particles of natural PPV by transmission electron microscopy, and VP2-VP22 does not observe the virus like particles in VP2-VP22, indicating that the N end fusion VP22 of VP2 does not affect the characteristics of VP2 to form the disease poison particles, and the C end fusion of exogenous proteins may be seen. The initial evaluation of the immunogenicity of VP2, VP22 short peptide and porcine parvovirus particles was preliminarily purified by the method of saturated ammonium sulfate to purify viral particles VP22-VP2 (VLPs-VP22-VP2), virus like particles VP2 (VLPs-VP2) and recombinant protein VP2-VP22, which were immunized with Freund's adjuvant, ISA 206 adjuvant and white oil adjuvant, respectively. The mice were compared with the PPV oil emulsion inactivated vaccine and PBS. The immunogenicity of the three recombinant proteins and the screening of the better immune adjuvant were compared and analyzed. The specific antibody of PPV in the serum of mice was detected by indirect ELISA. The results showed that VLPs-VP22-VP2 and VLPs-VP2 could effectively induce specific PPV antibodies in mice, and they were matched with ISA 206 adjuvant immunogenicity. The level of antibody against PPV inactivated vaccine was basically equal to that of PPV inactivated vaccine, while the level of antibody produced by recombinant protein VP2-VP22 was significantly lower than that in PPV inactivated vaccine group; IL-2 was the main growth factor of T cell proliferation, and IFN- gamma was produced only by activated T cells and natural killer cells (NK cells). The detection of these two indexes could reflect the cellular immunity of the animal body. The levels of serum IL-2 and IFN- gamma in the collected immunized mice showed that, compared with the negative control group, the VLPs-VP22-VP2, VLPs-VP2 and the recombinant protein VP2-VP22 immunization groups could effectively stimulate the production of IL-2 and IFN- gamma, and the VLPs-VP22-VP2 stimulation of VP22's VLPs-VP22-VP2 stimulates IFN- y and IL-2 was significantly higher than that of VLPs-VP2 and inactivated vaccine. (P0.05), in addition, with ISA 206 as an adjuvant, its immune enhancement effect was better than other adjuvant (P0.05). The results showed that the fusion of VP22 protein could obviously improve the immune response level of the specific T lymphocyte of VP2 protein, but it could not significantly improve the humoral immune effect of VP2 protein, and the immune effect of VP2N terminal fusion VP22 protein was better than that of it. C terminal fusion.VLPs-VP2 and VLPs-VP22-VP2 combined with ISA 206 adjuvant immunized mice could induce the body to produce humoral and cellular immune responses to PPV more effectively.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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