【摘要】：目前,豬繁殖與呼吸綜合征(PRRS)在全球范圍內(nèi)流行,基因型表現(xiàn)出高度可變性,對(duì)疾病的防控產(chǎn)生了巨大的阻礙�，F(xiàn)階段對(duì)PRRS的防控主要采取疫苗接種的方式,但存在一定的局限性,現(xiàn)有疫苗不能對(duì)豬群提供全面持久的保護(hù),因此積極發(fā)展新型疫苗對(duì)控制PRRSV感染與流行有著重要的意義。自成功采用乙肝病毒核心蛋白(HBc)作為疫苗載體表達(dá)了口蹄疫病毒抗原表位以來,HBc被越來越多的應(yīng)用于病毒樣顆粒(VLPs)疫苗的研制。作為VLPs疫苗載體,HBc具有以下特點(diǎn):天然條件下能夠自我組裝成顆粒樣結(jié)構(gòu),插入位點(diǎn)靈活,裝配效率高(95%),可以提呈抗原,能夠在多種表達(dá)系統(tǒng)中穩(wěn)定表達(dá)等。本研究以HBc為載體表達(dá)PRRSV GP5保護(hù)性抗原,構(gòu)建了HBc-GP5表位VLPs候選疫苗,并對(duì)其進(jìn)行小鼠免疫評(píng)價(jià)。首先根據(jù)Escherichia coli(strain K12)密碼子偏好性對(duì)HBc序列進(jìn)行密碼子優(yōu)化,并將其克隆至改造的pET-28a原核表達(dá)載體中,獲得重組質(zhì)粒pEO-NEW。經(jīng)SDS-PAGE、Western blot及透射電鏡等方法對(duì)表達(dá)獲得的VLPs進(jìn)行鑒定,結(jié)果表明成功誘導(dǎo)表達(dá)了HBc蛋白且表達(dá)后蛋白能夠自主裝配成VLPs,為下一步重組疫苗制備提供研究基礎(chǔ)。基于所得到的pEO-NEW表達(dá)載體,將優(yōu)化后的GP5基因插入至pEO-NEW載體的免疫顯性區(qū)(MIR),獲得重組質(zhì)粒pEO-LEORF。通過SDS-PAGE和Western blot方法鑒定重組目的蛋白,結(jié)果顯示,成功誘導(dǎo)表達(dá)GP5蛋白。經(jīng)透射電鏡對(duì)其形態(tài)進(jìn)行觀察,結(jié)果顯示,誘導(dǎo)后的重組蛋白能夠自主裝配成VLPs。以小鼠為模型分析該HBc-GP5 VLPs候選疫苗的免疫原性,利用ELISA進(jìn)行抗體檢測,結(jié)果表明其能夠誘導(dǎo)機(jī)體產(chǎn)生較高的特異抗體水平;對(duì)T細(xì)胞亞群及細(xì)胞因子進(jìn)行檢測結(jié)果表明,疫苗免疫組小鼠CD3+CD4+T細(xì)胞淋巴細(xì)胞亞型數(shù)量及IL-4水平顯著高于對(duì)照組(P0.05),淋巴細(xì)胞刺激指數(shù)(SI)及第三次免疫后IFN-γ含量與對(duì)照組相比顯著提高(P0.05),說明HBc-GP5表位VLPs候選疫苗能夠模擬病毒樣顆粒,有效提呈GP5并誘導(dǎo)機(jī)體產(chǎn)生體液和細(xì)胞免疫,為下一步實(shí)驗(yàn)研究提供數(shù)據(jù)基礎(chǔ)。
[Abstract]:At present, porcine reproductive and respiratory syndrome (PRRS) is prevalent all over the world, and the genotypes exhibit high variability, which is a great hindrance to the prevention and control of swine reproductive and respiratory syndrome (RRS). At present, vaccination is mainly used to prevent and control PRRS, but there are some limitations. The existing vaccine can not provide comprehensive and lasting protection for swine population. Therefore, it is of great significance to develop new vaccine to control PRRSV infection and epidemic. Since hepatitis B virus core protein (HBc) was successfully used as vaccine vector to express the antigen epitopes of foot-and-mouth disease virus, HBc has been used more and more in the development of virus-like granular (VLPs) vaccine. As a vector of VLPs vaccine, HBc has the following characteristics: it can self-assemble into granular structure under natural conditions, the insertion site is flexible, the assembly efficiency is high (95%), it can present antigen, and can be expressed stably in many expression systems. In this study, HBc was used as vector to express PRRSV GP5 protective antigen, and the candidate vaccine of HBc-GP5 epitope VLPs was constructed and evaluated in mice. Firstly, according to the Escherichia coli (strain K12) codon preference, the HBc sequence was optimized and cloned into the modified pET-28a prokaryotic expression vector to obtain the recombinant plasmid pEO-NEW.. The expression of VLPs was identified by SDS-PAGE,Western blot and transmission electron microscopy. The results showed that the expression of HBc protein was successfully induced and the expressed protein could be assembled into VLPs, by itself to provide the basis for further preparation of recombinant vaccine. Based on the obtained pEO-NEW expression vector, the optimized GP5 gene was inserted into the immune dominant region (MIR), of the pEO-NEW vector to obtain the recombinant plasmid pEO-LEORF.. The recombinant target protein was identified by SDS-PAGE and Western blot. The results showed that the expression of GP5 protein was successfully induced. The morphology of the recombinant protein was observed by transmission electron microscope. The results showed that the induced recombinant protein could assemble into VLPs. by itself. The immunogenicity of the HBc-GP5 VLPs vaccine was analyzed by using the mouse model and the antibody was detected by ELISA. The results showed that the vaccine could induce the body to produce a higher level of specific antibody. The detection of T cell subsets and cytokines showed that the number of CD3 CD4 T lymphocyte subtypes and the level of IL-4 in the vaccine immunized mice were significantly higher than those in the control group (P0.05). The lymphocyte stimulating index (SI) and the content of IFN- 緯 after the third immunization were significantly higher than those in the control group (P0.05), which indicated that the candidate vaccine of HBc-GP5 epitope VLPs could mimic virus-like particles. Effectively presenting GP5 and inducing humoral and cellular immunity provide data basis for further experimental research.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S855.3

【共引文獻(xiàn)】

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本文編號(hào)：2371525