本文選題：桿狀病毒 + 表面展示��； 參考：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：與其他的病毒載體系統(tǒng)比較,桿狀病毒用于基因轉(zhuǎn)移和疫苗遞送擁有顯著的優(yōu)勢,如插入外源片段容量大、操作簡單、生物安全性高(不具有整合性和哺乳動物致病性)以及哺乳動物中不存在針對桿狀病毒的先天免疫。攜帶哺乳動物病毒活性啟動子的重組桿狀病毒(BacMam virus)用于哺乳動物細(xì)胞的轉(zhuǎn)基因而被廣泛研究。然而,桿狀病毒用于哺乳動物的基因轉(zhuǎn)移仍面臨兩大瓶頸:哺乳動物補(bǔ)體系統(tǒng)的清除和哺乳動物細(xì)胞轉(zhuǎn)導(dǎo)效率低,尤其是針對樹突細(xì)胞(DCs)、巨噬細(xì)胞(MФ)、B細(xì)胞和T細(xì)胞等免疫細(xì)胞表現(xiàn)出極低的轉(zhuǎn)導(dǎo)效率。這在一定程度上限制了桿狀病毒載體疫苗的效力。本實(shí)驗(yàn)室前期研究發(fā)現(xiàn),表面展示豬IgG1 Fc可通過增強(qiáng)桿狀病毒補(bǔ)體逃逸和轉(zhuǎn)導(dǎo)哺乳動物細(xì)胞從而提高桿狀病毒載體疫苗的效力。IgG Fc在體內(nèi)主要通過轉(zhuǎn)運(yùn)途徑(FcRn)、補(bǔ)體途徑(C1q)和免疫細(xì)胞識別途徑(FcγRI)來調(diào)控誘導(dǎo)免疫反應(yīng)�？紤]到豬不同亞型IgG Fc在結(jié)構(gòu)上存在明顯差異,在與上述三種途徑識別受體的結(jié)合能力方面也存在差異。本研究通過嘗試構(gòu)建表面展示不同亞型IgG Fc的桿狀病毒,探究不同亞型IgG Fc是否具有類似IgG1 Fc的功能,并篩選更具佐劑潛力的IgG Fc亞型,為構(gòu)建高效的拮抗補(bǔ)體重組桿狀病毒疫苗載體提供理論支持和技術(shù)手段。本研究取得了以下結(jié)果:1.重組桿狀病毒的構(gòu)建和鑒定本試驗(yàn)基于MultiBac桿狀病毒表達(dá)系統(tǒng),通過在載體pFBDM-P10-gp64SP-vsvgED多克隆位點(diǎn)中插入不同亞型IgG Fc構(gòu)建桿狀病毒轉(zhuǎn)移載體,經(jīng)脂質(zhì)體介導(dǎo)的方法轉(zhuǎn)染Sf9昆蟲細(xì)胞包裝重組桿狀病毒。Western blot和間接免疫熒光分析顯示目的基因正確正確表達(dá)并定位在重組桿狀病毒表面。這些結(jié)果表明,成功構(gòu)建了8種展示不同亞型IgG Fc的VSV-G假型化的重組桿狀病毒。2.重組桿狀病毒的豬血清補(bǔ)體系統(tǒng)拮抗能力和體外轉(zhuǎn)基因表達(dá)評價(jià)通過體外試驗(yàn)比較展示不同亞型IgG Fc的重組桿狀病毒在豬血清中的存活率,與對照病毒BV-vsvg相比(16.59%),展示豬不同亞型IgG Fc的重組桿狀病毒存活率均顯著提高,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的重組病毒(BV-vsvg-hFc和BV-vsvg-rhFc)存活率(17.04%~23.9%)與對照病毒沒有顯著差異。轉(zhuǎn)導(dǎo)IEC細(xì)胞后,轉(zhuǎn)基因表達(dá)結(jié)果顯示,展示豬IgG Fc不同亞型的重組桿狀病毒在轉(zhuǎn)導(dǎo)效率和轉(zhuǎn)基因表達(dá)上較對照病毒均有顯著增強(qiáng),其中BV-vsvg-pFc3最高,其次是BV-vsvg-rpFc3、BV-vsvg-pFc2和BV-vsvg-pFc。而由于種屬性差異,展示人IgG1 Fc的重組桿狀病毒BV-vsvg-hFc和BV-vsvg-rhFc在轉(zhuǎn)基因表達(dá)水平上與對照病毒BV-vsvg均無顯著差異。3.展示Fc重組桿狀病毒與Fc激活型受體FcγRI結(jié)合能力比較通過構(gòu)建FcγRI表達(dá)載體,成功表達(dá)和純化了豬IgG Fc激活型受體FcγRI。選取在補(bǔ)體逃逸和轉(zhuǎn)基因表達(dá)兩個(gè)方面都有良好表現(xiàn)的3株重組桿狀病毒,以重組FcγRI作為包被物,通過ELISA分析比較其與FcγRI的結(jié)合能力,其中BV-vsvg-pFc3與FcγRI結(jié)合能力最強(qiáng),其次是BV-vsvg-pFc2和BV-vsvg-pFc。綜上,本研究構(gòu)建了8種展示不同亞型IgG Fc的重組桿狀病毒,補(bǔ)體拮抗能力、體外轉(zhuǎn)基因表達(dá)和與激活型受體FcγRI結(jié)合活性三個(gè)評價(jià)指標(biāo)顯示,表面展示豬IgG3 Fc(pFc3)的重組桿狀病毒用于豬用載體疫苗的研發(fā)更具發(fā)展?jié)摿Α?br/>[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.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.4

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本文編號：1855247