表面展示猪不同亚型IgG Fc的重组杆状病毒疫苗载体研究

发布时间：2018-05-07 03:35

本文选题：杆状病毒 + 表面展示　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：与其他的病毒载体系统比较,杆状病毒用于基因转移和疫苗递送拥有显著的优势,如插入外源片段容量大、操作简单、生物安全性高(不具有整合性和哺乳动物致病性)以及哺乳动物中不存在针对杆状病毒的先天免疫。携带哺乳动物病毒活性启动子的重组杆状病毒(BacMam virus)用于哺乳动物细胞的转基因而被广泛研究。然而,杆状病毒用于哺乳动物的基因转移仍面临两大瓶颈:哺乳动物补体系统的清除和哺乳动物细胞转导效率低,尤其是针对树突细胞(DCs)、巨噬细胞(MФ)、B细胞和T细胞等免疫细胞表现出极低的转导效率。这在一定程度上限制了杆状病毒载体疫苗的效力。本实验室前期研究发现,表面展示猪IgG1 Fc可通过增强杆状病毒补体逃逸和转导哺乳动物细胞从而提高杆状病毒载体疫苗的效力。IgG Fc在体内主要通过转运途径(FcRn)、补体途径(C1q)和免疫细胞识别途径(FcγRI)来调控诱导免疫反应。考虑到猪不同亚型IgG Fc在结构上存在明显差异,在与上述三种途径识别受体的结合能力方面也存在差异。本研究通过尝试构建表面展示不同亚型IgG Fc的杆状病毒,探究不同亚型IgG Fc是否具有类似IgG1 Fc的功能,并筛选更具佐剂潜力的IgG Fc亚型,为构建高效的拮抗补体重组杆状病毒疫苗载体提供理论支持和技术手段。本研究取得了以下结果:1.重组杆状病毒的构建和鉴定本试验基于MultiBac杆状病毒表达系统,通过在载体pFBDM-P10-gp64SP-vsvgED多克隆位点中插入不同亚型IgG Fc构建杆状病毒转移载体,经脂质体介导的方法转染Sf9昆虫细胞包装重组杆状病毒。Western blot和间接免疫荧光分析显示目的基因正确正确表达并定位在重组杆状病毒表面。这些结果表明,成功构建了8种展示不同亚型IgG Fc的VSV-G假型化的重组杆状病毒。2.重组杆状病毒的猪血清补体系统拮抗能力和体外转基因表达评价通过体外试验比较展示不同亚型IgG Fc的重组杆状病毒在猪血清中的存活率,与对照病毒BV-vsvg相比(16.59%),展示猪不同亚型IgG Fc的重组杆状病毒存活率均显著提高,其中BV-vsvg-pFc2(52%)最高,其次是BV-vsvg-pFc3(49.39%)和BV-vsvg-pFc(47.2%)。而展示人IgG1 Fc的重组病毒(BV-vsvg-hFc和BV-vsvg-rhFc)存活率(17.04%~23.9%)与对照病毒没有显著差异。转导IEC细胞后,转基因表达结果显示,展示猪IgG Fc不同亚型的重组杆状病毒在转导效率和转基因表达上较对照病毒均有显著增强,其中BV-vsvg-pFc3最高,其次是BV-vsvg-rpFc3、BV-vsvg-pFc2和BV-vsvg-pFc。而由于种属性差异,展示人IgG1 Fc的重组杆状病毒BV-vsvg-hFc和BV-vsvg-rhFc在转基因表达水平上与对照病毒BV-vsvg均无显著差异。3.展示Fc重组杆状病毒与Fc激活型受体FcγRI结合能力比较通过构建FcγRI表达载体,成功表达和纯化了猪IgG Fc激活型受体FcγRI。选取在补体逃逸和转基因表达两个方面都有良好表现的3株重组杆状病毒,以重组FcγRI作为包被物,通过ELISA分析比较其与FcγRI的结合能力,其中BV-vsvg-pFc3与FcγRI结合能力最强,其次是BV-vsvg-pFc2和BV-vsvg-pFc。综上,本研究构建了8种展示不同亚型IgG Fc的重组杆状病毒,补体拮抗能力、体外转基因表达和与激活型受体FcγRI结合活性三个评价指标显示,表面展示猪IgG3 Fc(pFc3)的重组杆状病毒用于猪用载体疫苗的研发更具发展潜力。
[Abstract]:Compared with other viral vector systems, baculovirus has significant advantages for gene transfer and vaccine delivery, such as large capacity, simple operation, high biosecurity (without integration and mammalian pathogenicity), and innate immunity to baculovirus in mammals, and mammalian viruses. The recombinant baculovirus (BacMam virus) of active promoter is widely used in mammalian cells. However, the transfer of baculovirus for mammalian gene transfer is still facing two major bottlenecks: the removal of mammalian complement system and the low efficiency of mammalian cell transduction, especially for dendritic cells (DCs), macrophages (M) B cells and T cells show very low transduction efficiency. This restricts the effectiveness of baculovirus vector vaccines to some extent. Earlier studies in our laboratory found that the surface display of porcine IgG1 Fc could improve the potency of baculovirus vector vaccine by enhancing baculovirus complement and transduction of mammalian cells from.IgG Fc. In vivo mainly through the transport pathway (FcRn), complement pathway (C1q) and immune cell recognition pathway (Fc gamma RI) to regulate the induction of immune response. Considering that different subtypes of IgG Fc have distinct structural differences, and there are differences in the binding capacity of the three ways to identify the receptors. The baculovirus of subtype IgG Fc, exploring whether different subtypes of IgG Fc have the function of IgG1 Fc, and screening the IgG Fc subtype with the potential of adjuvant, provide theoretical support and technical means for the construction of effective antagonistic complement recombinant baculovirus vector. The following results have been obtained: 1. the construction and identification of recombinant baculovirus Based on the MultiBac baculovirus expression system, baculovirus transfer vectors were constructed by inserting different subtypes of IgG Fc into the vector pFBDM-P10-gp64SP-vsvgED polyclonal sites. The transfection of Sf9 insect cell packaging recombinant baculovirus.Western blot by liposome mediated method and indirect immunofluorescence analysis showed that the target gene was correct and correct. The results showed that 8 kinds of VSV-G false recombinant baculovirus.2. recombinant baculovirus.2. recombinant baculovirus with different subtypes of IgG Fc was successfully constructed and the gene expression evaluation of recombinant baculovirus in vitro and in vitro transgenic expression evaluation showed that the recombinant baculovirus of different subtypes of IgG Fc was displayed in vitro. The survival rate in the pig serum was compared with the control virus BV-vsvg (16.59%). The survival rate of recombinant baculovirus showing the different subtypes of IgG Fc in pigs was significantly increased, of which BV-vsvg-pFc2 (52%) was the highest, followed by BV-vsvg-pFc3 (49.39%) and BV-vsvg-pFc (47.2%). The survival rate of the recombinant virus (BV-vsvg-hFc and BV-vsvg-rhFc) of IgG1 Fc (17.04%~23.9) was shown (17.04%~23.9) There was no significant difference between the control virus and the control virus. After transduction of IEC cells, the transgenic expression showed that the recombinant baculovirus showing the different subtypes of the pig IgG Fc was significantly enhanced in the transduction efficiency and the transgenic expression, of which BV-vsvg-pFc3 was the highest, followed by BV-vsvg-rpFc3, BV-vsvg-pFc2 and BV-vsvg-pFc. due to the poor seed property. The recombinant baculovirus BV-vsvg-hFc and BV-vsvg-rhFc of IgG1 Fc showed no significant difference from the control virus BV-vsvg at the transgenic expression level,.3. showed the binding capacity of the Fc recombinant baculovirus and Fc activating receptor Fc gamma RI by constructing the Fc RI expression vector. 3 recombinant baculovirus, which have good performance in two aspects of complement escape and gene expression, were recombined with recombinant Fc gamma RI as a clad, and compared with Fc gamma RI by ELISA analysis. The combination of BV-vsvg-pFc3 and Fc gamma RI was the strongest, followed by BV-vsvg-pFc2 and BV-vsvg-pFc., and 8 different subtypes were constructed. The recombinant baculovirus of type IgG Fc, complement antagonism, in vitro transgene expression and the binding activity of Fc gamma RI with activator receptor show that the recombinant baculovirus on the surface of porcine IgG3 Fc (pFc3) is more potential for the development of porcine carrier vaccine.

【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.4

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