细胞内表达抗独特型单链抗体抑制猪繁殖与呼吸综合征病毒感染MARC-145细胞的研究

发布时间：2018-01-19 19:28

本文关键词： 抗独特型抗体猪繁殖与呼吸综合征病毒单链抗体　出处：《西北农林科技大学》2015年博士论文　论文类型：学位论文

【摘要】：猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)为单股正链RNA病毒,与鼠乳酸脱氢酶增高病毒、马动脉炎病毒和猴出血热病毒同属套氏病毒目动脉炎病毒科,是全球性猪传染病—猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)的病原。该病以母猪发生繁殖障碍、呼吸道疾病、哺乳仔猪的高热和高死亡率、免疫抑制和持续性感染等为主要特征。由于PRRSV表现出较其他RNA病毒更高的突变率,同时具有破坏宿主固有免疫系统的潜能,且迄今为止尚无有效的弱毒苗或灭活疫苗控制该病的发生与传播,PRRS几乎在世界上所有的养猪国家均有流行,造成了巨大的经济损失。本实验室早期研究证明PRRSV感染可产生针对GP5蛋白的抗独特型抗体,该抗体在机体抗病毒免疫应答及疾病转归中发挥重要作用;并且通过顺序免疫法得到了在功能上模拟PRRSV GP5蛋白的单克隆抗独特型抗体Mab2-5G2,研究证实该抗体仍保留自身抗独特型抗体的特性并且可下调弱毒苗对猪只的免疫保护作用。前期研究结果提示抗独特型抗体的功能较为复杂,其调控机体免疫应答的机制尚需要深入研究。然而,通过猪血清获得抗独特型抗体的数量有限且动物试验花费较大耗力较多,因此,构建细胞内表达单链抗体的细胞模型用于抗独特型抗体功能和免疫机制的前期研究更为理想。本论文的主要研究目的是构建抗独特型单链抗体的真核表达载体,经慢病毒转导使其在MARC-145细胞中表达,通过研究胞内表达抗独特型抗体后PRRSV的复制和增殖情况,验证其对病毒感染的影响并初步探索其产生机制。本课题研究的内容和结果如下:1.通过RT-PCR方法从Mab2-5G2杂交瘤细胞中分别扩增出重链可变区和轻链可变区基因,测序正确后通过弹性连接子(GGGGS)3经Overlap PCR连接组成一个完整的单链可变区抗体(scFv);再用GPGP连接子将5G2scFv和EGFP融合表达并连接至慢病毒表达载体;以抗禽戊型肝炎病毒E抗原单克隆抗体1B5为模板,构建1B5scFv作为对照,构建质粒分别命名为pTRIP-CMV-5G2scFv-GPGP-EGFP-IRES-Puro和pTRIP-CMV-1B5scFv-GPGP-EGFP-IRES-Puro。测序结果证实慢病毒载体构建成功。2.使用293T细胞包装重组慢病毒,收集细胞上清将表达5G2scFv和1B5scFv的重组慢病毒转导MARC-145细胞,经嘌呤霉素抗性筛选和增强绿色荧光蛋白(EGFP)筛选单克隆。间接免疫荧光试验和Western blot试验证实5G2scFv或1B5scFv在MARC-145细胞中稳定表达,细胞系构建成功,分别命名为MARC-5G2scFv和MARC-1B5scFv。细胞活力试验结果提示细胞内表达单链抗体不影响细胞活力。3.为了验证细胞内表达5G2scFv是否抑制PRRSV在MARC-145细胞中的复制增殖,本研究对MARC-5G2scFv细胞系进行了攻毒试验。SD16和VR-2332毒株感染48h后,通过Western blot和TCID50方法分别检测N蛋白和子代病毒的产生。试验结果显示N蛋白表达量在MARC-5G2scFv细胞中显著下降,而在MARC-1B5scFv细胞和MARC-145中不受影响;与上述发现一致,同一时间MARC-5G2scFv上清中的病毒滴度也显著降低(P0.05)。而且,该抑制作用在0.01MOI、0.1MOI和1MOI的接毒剂量时均可出现。接毒后不同时间收集上清并测定各组样品毒价后提示MARC-5G2sc Fv细胞上清中病毒滴度显著低于MARC-1B5scFv细胞和MARC-145细胞(P0.05),且在接毒后12h最为明显(P0.001)。4.通过病毒吸附试验和检测I型干扰素表达初步探讨胞内表达5G2scFv下调病毒复制增殖的机制。荧光定量PCR和病毒滴度检测结果显示病毒吸附并无明显改变(P0.05),提示胞内表达5G2scFv不影响病毒吸附MARC-145细胞。I型干扰素转录和表达检测结果显示,接毒48h后,MARC-5G2scFv细胞中IFN-α的转录和表达较未感染的MARC-5G2scFv细胞及感染后MARC-1B5scFv和MARC-145细胞明显升高(P0.05),而在MARC-1B5scFv和MARC-145细胞中无明显变化(P0.05);但是三组细胞中IFN-β的转录和表达在感染前后均无明显改变(P0.05)。以上结果提示胞内表达5G2scFv抑制病毒感染可能与IFN-α表达上调有关。综上所述,本研究通过构建抗独特型单链抗体并使其在MARC-145细胞中稳定表达,对5G2scFv胞内表达会否影响的PRRSV感染及其机制进行了一系列的研究。本试验首次构建稳定表达抗独特型单链抗体的MARC-145细胞系;证明其胞内表达可抑制病毒复制增殖;该作用并非通过抑制病毒吸附实现而与IFN-α分泌增加相关。本研究拓展了抗独特型单链抗体的应用,为寻找新的治疗方法和疫苗提供了新思路。
[Abstract]:Porcine reproductive and respiratory syndrome virus (porcine reproductive and respiratory syndrome virus, PRRSV) is a single stranded RNA virus and mouse lactate dehydrogenase increased virus, equine arteritis virus and simian virus are nested virales Arteriviridae, is the global swine infectious disease, porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS). The pathogen of the disease in order to sow reproductive failure, respiratory disease, high fever and high mortality of piglets, immune suppression and persistent infection as the main feature. By PRRSV showed higher than that of other RNA virus mutation rate, at the same time with the destruction of the host innate immune system and potential. So far there is no effective vaccine or inactivated vaccine to control the occurrence and spread of the disease, PRRS in almost all the world's countries have pig epidemic, caused great The economic loss of the laboratory. Early studies have proved that PRRSV infection can produce the anti idiotypic antibody of GP5 protein, the antibodies play an important role in the outcome of antiviral immune response and disease; and the monoclonal analog PRRSV GP5 protein in the function of the anti idiotypic antibody Mab2-5G2 were obtained by sequential immunization, the antibody is proved the retention characteristics of anti idiotypic antibodies and can downregulate the immune protective effect of vaccine for pigs. The previous research results suggest that anti idiotypic antibody complex functions still need in-depth study in the regulation of the immune response mechanism. However, the limited number of pig serum obtained and animal experiments of anti idiotypic antibodies to spend large consumption more, therefore, for the construction of preliminary study on anti idiotypic antibody and immune function mechanism of cell model for the single chain antibody expression in vitro Ideal. The main purpose of this study is to construct eukaryotic expression vector of anti idiotypic antibody by lentivirus transduction, the expression in MARC-145 cells, the expression of replication and proliferation of PRRSV anti idiotypic antibody by investigating the intracellular viral infection to verify its effect and explore the mechanism this topic. The contents and results are as follows: 1. by the RT-PCR method from the Mab2-5G2 hybridoma cells were amplified by the variable region genes of heavy and light chain variable region, after sequencing by elastic linker (GGGGS) 3 by Overlap PCR are connected to form a complete scFv (scFv); and the GPGP connector 5G2scFv and EGFP fusion expression and connected to the lentiviral expression vector; anti avian hepatitis E virus E antigen monoclonal antibody 1B5 as template to construct 1B5scFv as control plasmid named pTRIP-CMV-5G2 ScFv-GPGP-EGFP-IRES-Puro and pTRIP-CMV-1B5scFv-GPGP-EGFP-IRES-Puro. sequencing results confirmed that lentiviral vectors using 293T cells to package the recombinant lentivirus successfully.2. collected supernatant 5G2scFv expression and 1B5scFv recombinant lentiviral transduction of MARC-145 cells by puromycin resistance screening and enhanced green fluorescent protein (EGFP) monoclonal screening. Indirect immunofluorescence test and Western test showed that 5G2scFv or blot stable expression of 1B5scFv in MARC-145 cells, cell lines were constructed successfully, which were named MARC-5G2scFv and MARC-1B5scFv. cell viability test indicate that the antibody did not affect cell viability of.3. cells in order to verify the expression of replication of 5G2scFv whether inhibition of PRRSV expression in MARC-145 cells in the cell, this study conducted a challenge test with.SD16 and VR-2332 strains of 48h infection the MARC-5G2scFv cell line, by Western blo T and TCID50 were used to detect N protein and progeny virus production. Test results showed that the expression of N protein in MARC-5G2scFv cells decreased significantly in MARC-1B5scFv cells and MARC-145 were not affected; found consistent with the above, the same time MARC-5G2scFv in the supernatant of virus titer was significantly lower (P0.05). Also, the inhibitory effect on 0.01MOI, inoculation dose can be 0.1MOI and 1MOI. The supernatant was collected at different time after inoculation and determination of each sample after virus titer indicated that the virus titer in the supernatant of MARC-5G2sc Fv cells was significantly lower than that of MARC-1B5scFv cells and MARC-145 cells (P0.05), and the most obvious 12h after inoculation (P0.001).4. to investigate the proliferation mechanism of down-regulation of 5G2scFv virus replication by intracellular expression of virus adsorption test and detection of type I interferon expression. Fluorescence quantitative PCR and virus titer showed no significant change in virus adsorption and Change (P0.05), suggesting that 5G2scFv does not affect the virus adsorption and the expression of MARC-145 cell type.I interferon transcription assay showed the expression of intracellular 48h after inoculation, MARC-1B5scFv and MARC-145 cells significantly increased in MARC-5G2scFv cells and MARC-5G2scFv cells infected with IFN- alpha in transcription and expression than those without infection after (P0.05), but no significant change in MARC-1B5scFv and MARC-145 cells (P0.05); but the transcription and expression of IFN- in beta cells of three groups in before and after infection showed no significant change (P0.05). These results suggest that intracellular expression of 5G2scFv and IFN- could inhibit the virus infection of alpha expression. In summary, this study builds the anti idiotypic antibody and its stability the expression of 5G2scFv in MARC-145 cells, intracellular expression of PRRSV infection and its mechanism will affect conducted a series of studies. It is the first time to build a stable expression of anti anti idiotypic scFv MARC-145 cell body; that can inhibit viral replication and proliferation of the intracellular expression; the effect is not through the inhibition of virus adsorption and increased secretion and IFN- alpha. This study extends the application of anti idiotypic antibody, and provides a new way to find new treatments and vaccines.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S858.28

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