本文選題：豬細小病毒 + VP2蛋白��； 參考：《廣西大學》2017年碩士論文

【摘要】：豬細小病毒(Porcine parvovirus,PPV)是引起母豬繁殖障礙的主要病原之一,疫苗免疫仍是預(yù)防控制該病的主要手段。病毒樣粒子(Virus-like particles,VLPs)疫苗以其安全性高、免疫原性好成為各類病毒疫苗研究的熱門方向。PPV病毒樣粒子(PPV-VLPs)是不含PPVDNA的空衣殼結(jié)構(gòu),由PPVVP2(下文簡稱:VP2)結(jié)構(gòu)蛋白體外自行組裝形成,形態(tài)上與天然病毒粒子相似,具有很強的免疫原性和生物學活性。單純皰疹病毒1型(HSV-1)VP22蛋白是該病毒的結(jié)構(gòu)蛋白,不僅具有細胞間自由轉(zhuǎn)運的能力,而且還可以促進融合抗原交叉致敏,增強免疫原性。此外,HSV-1VP22(下文簡稱:VP22)蛋白還可能提高DNA疫苗的免疫反應(yīng)。本研究分為三個部分,詳細研究內(nèi)容如下:1、細胞穿膜肽VP22對生物膜穿透性的研究以載體pEGFP-N1為模板擴增增強型綠色熒光蛋白(EGFP)基因,通過SOE-PCR分別將VP22基因片段拼接到EGFP基因的3'/5'端,獲得VP22-EGFP和EGFP-VP22融合基因;將融合基因及EGFP基因分別克隆到pET-28a(+)載體構(gòu)建重組表達質(zhì)粒 pET28a-EGFP-VP22、pET28a-VP22-EGFP和pET28a-EGFP,并進行測序分析及酶切鑒定,將鑒定正確的各重組表達質(zhì)粒及pET-28a空載體分別轉(zhuǎn)化宿主菌E.coli BL21(DE3)進行表達,表達產(chǎn)物經(jīng)SDS-PAGE及Western Blot分析結(jié)果可見相對分子量分別為約31 kDa、31 kDa及27kDa的蛋白分子條帶,大小與預(yù)期相符,表明EGFP-VP22、VP22-EGFP、EGFP蛋白表達成功。對表達的蛋白進行Ni-NTA親和層析純化,將純化后的蛋白添加到Vero細胞中進行共培養(yǎng),l0h后熒光顯微鏡下觀察到加入VP22-EGFP和EGFP-VP22蛋白的細胞有綠色熒光,而加入EGFP的細胞與空白對照組細胞中則沒有觀察到綠色熒光存在,提示細胞穿膜肽VP22可有效攜帶與其融合的EGFP蛋白穿過細胞膜進入細胞,VP22蛋白融合到EGFP蛋白N端或C端并不影響VP22的穿膜效果。2、VP22短肽融合豬細小病毒樣粒子的制備及鑒定利用Bac-to-Bac桿狀病毒表達載體系統(tǒng)進行PPV VLPs的制備。以PPV N株的基因組為模板擴增其VP2基因,通過SOE-PCR分別VP22基因拼接到VP2基因片段的3'/5'端,獲得VP22-VP2和VP2-VP22融合基因;將兩種融合基因及VP2基因分別克隆到pFastBacTMl載體,獲得重組質(zhì)粒pFB-VP22-VP2、pFB-VP2-VP22 及 pFB-VP2。重組質(zhì)粒轉(zhuǎn)化至 DHlOBac 感受態(tài)細胞進行同源重組,獲得重組桿粒Bacmid-VP22-VP2、Bacmid-VP2-VP22及Bacmid-VP2。重組桿粒經(jīng)脂質(zhì)體法轉(zhuǎn)染sf9細胞,獲得重組桿狀病毒株rBac-VP22-VP2、rBac-VP2-VP22及rBac-VP2。隨后將重組桿狀病毒株感染到sf9細胞進行重組蛋白的表達,表達產(chǎn)物經(jīng)SDS-PAGE和Western-blot鑒定分析,結(jié)果可見相對分子量分別為約67 kDa、67 kDa及64 kDa的蛋白分子條帶,大小與預(yù)期相符,表明VP2-VP22、VP22-VP2及VP2蛋白表達成功。進一步用PPV單抗對重組桿狀病毒感染細胞進行IFA鑒定,可觀察到特異性熒光,再次證實重組蛋白的表達且具有生物活性。表達產(chǎn)物經(jīng)透射電鏡觀察,VP2和VP22-VP2均可自我組裝形成22-24nm、形態(tài)類似天然PPV的病毒樣粒子;VP2-VP22沒有觀察到病毒樣粒子,表明VP2的N端融合VP22不影響VP2形成病毒粒子的特性,而C端融合外源蛋白可能影響VP2形成病毒樣粒子。3、VP22短肽融合豬細小病毒樣粒子免疫原性的初步評價用飽和硫酸銨法初步純化病毒樣粒子VP22-VP2(VLPs-VP22-VP2)、病毒樣粒子VP2(VLPs-VP2)及重組蛋白VP2-VP22,分別配以弗氏佐劑、ISA 206佐劑及白油司本佐劑制成疫苗免疫小鼠,并以PPV油乳滅活苗和PBS為對照,比較分析三種重組蛋白的免疫原性及篩選較好的免疫佐劑。通過間接ELISA檢測免疫小鼠血清中PPV特異性抗體,結(jié)果顯示VLPs-VP22-VP2及VLPs-VP2能有效誘導小鼠產(chǎn)生特異性PPV抗體,其配以ISA 206佐劑免疫產(chǎn)生的抗體水平與PPV滅活苗基本相當,而重組蛋白VP2-VP22產(chǎn)生的抗體水平要明顯低于PPV滅活苗組;IL-2是T細胞增殖的主要生長因子,IFN-γ僅由活化T細胞和自然殺傷細胞(NK細胞)產(chǎn)生,檢測這兩個指標的不同程度能反映動物機體的細胞免疫水平,對采集的免疫小鼠血清IL-2和IFN-γ含量測定結(jié)果顯示,與陰性對照組相比,VLPs-VP22-VP2、VLPs-VP2及重組蛋白VP2-VP22免疫組均能有效刺激IL-2和IFN-γ的產(chǎn)生,且融合VP22的VLPs-VP22-VP2刺激機體產(chǎn)生IFN-γ和IL-2的水平顯著高于VLPs-VP2及PPV滅活苗(P0.05),此外,以ISA 206為佐劑,其免疫增強的效果要優(yōu)于其他佐劑(P0.05)。上述結(jié)果表明,融合VP22蛋白可明顯提高VP2蛋白的特異性T淋巴細胞的免疫應(yīng)答水平,但卻不能顯著提高VP2蛋白的體液免疫效果;VP2N端融合VP22蛋白的免疫效果要優(yōu)于其C端融合。VLPs-VP2、VLPs-VP22-VP2配以ISA 206佐劑免疫小鼠后能更有效地誘導機體產(chǎn)生針對PPV的體液及細胞免疫。
[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.
【學位授予單位】：廣西大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.65

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