本文關(guān)鍵詞： 抗獨特型抗體 豬繁殖與呼吸綜合征病毒 單鏈抗體　出處：《西北農(nóng)林科技大學》2015年博士論文　論文類型：學位論文

【摘要】：豬繁殖與呼吸綜合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)為單股正鏈RNA病毒,與鼠乳酸脫氫酶增高病毒、馬動脈炎病毒和猴出血熱病毒同屬套氏病毒目動脈炎病毒科,是全球性豬傳染病—豬繁殖與呼吸綜合征(Porcine reproductive and respiratory syndrome,PRRS)的病原。該病以母豬發(fā)生繁殖障礙、呼吸道疾病、哺乳仔豬的高熱和高死亡率、免疫抑制和持續(xù)性感染等為主要特征。由于PRRSV表現(xiàn)出較其他RNA病毒更高的突變率,同時具有破壞宿主固有免疫系統(tǒng)的潛能,且迄今為止尚無有效的弱毒苗或滅活疫苗控制該病的發(fā)生與傳播,PRRS幾乎在世界上所有的養(yǎng)豬國家均有流行,造成了巨大的經(jīng)濟損失。本實驗室早期研究證明PRRSV感染可產(chǎn)生針對GP5蛋白的抗獨特型抗體,該抗體在機體抗病毒免疫應答及疾病轉(zhuǎn)歸中發(fā)揮重要作用;并且通過順序免疫法得到了在功能上模擬PRRSV GP5蛋白的單克隆抗獨特型抗體Mab2-5G2,研究證實該抗體仍保留自身抗獨特型抗體的特性并且可下調(diào)弱毒苗對豬只的免疫保護作用。前期研究結(jié)果提示抗獨特型抗體的功能較為復雜,其調(diào)控機體免疫應答的機制尚需要深入研究。然而,通過豬血清獲得抗獨特型抗體的數(shù)量有限且動物試驗花費較大耗力較多,因此,構(gòu)建細胞內(nèi)表達單鏈抗體的細胞模型用于抗獨特型抗體功能和免疫機制的前期研究更為理想。本論文的主要研究目的是構(gòu)建抗獨特型單鏈抗體的真核表達載體,經(jīng)慢病毒轉(zhuǎn)導使其在MARC-145細胞中表達,通過研究胞內(nèi)表達抗獨特型抗體后PRRSV的復制和增殖情況,驗證其對病毒感染的影響并初步探索其產(chǎn)生機制。本課題研究的內(nèi)容和結(jié)果如下:1.通過RT-PCR方法從Mab2-5G2雜交瘤細胞中分別擴增出重鏈可變區(qū)和輕鏈可變區(qū)基因,測序正確后通過彈性連接子(GGGGS)3經(jīng)Overlap PCR連接組成一個完整的單鏈可變區(qū)抗體(scFv);再用GPGP連接子將5G2scFv和EGFP融合表達并連接至慢病毒表達載體;以抗禽戊型肝炎病毒E抗原單克隆抗體1B5為模板,構(gòu)建1B5scFv作為對照,構(gòu)建質(zhì)粒分別命名為pTRIP-CMV-5G2scFv-GPGP-EGFP-IRES-Puro和pTRIP-CMV-1B5scFv-GPGP-EGFP-IRES-Puro。測序結(jié)果證實慢病毒載體構(gòu)建成功。2.使用293T細胞包裝重組慢病毒,收集細胞上清將表達5G2scFv和1B5scFv的重組慢病毒轉(zhuǎn)導MARC-145細胞,經(jīng)嘌呤霉素抗性篩選和增強綠色熒光蛋白(EGFP)篩選單克隆。間接免疫熒光試驗和Western blot試驗證實5G2scFv或1B5scFv在MARC-145細胞中穩(wěn)定表達,細胞系構(gòu)建成功,分別命名為MARC-5G2scFv和MARC-1B5scFv。細胞活力試驗結(jié)果提示細胞內(nèi)表達單鏈抗體不影響細胞活力。3.為了驗證細胞內(nèi)表達5G2scFv是否抑制PRRSV在MARC-145細胞中的復制增殖,本研究對MARC-5G2scFv細胞系進行了攻毒試驗。SD16和VR-2332毒株感染48h后,通過Western blot和TCID50方法分別檢測N蛋白和子代病毒的產(chǎn)生。試驗結(jié)果顯示N蛋白表達量在MARC-5G2scFv細胞中顯著下降,而在MARC-1B5scFv細胞和MARC-145中不受影響;與上述發(fā)現(xiàn)一致,同一時間MARC-5G2scFv上清中的病毒滴度也顯著降低(P0.05)。而且,該抑制作用在0.01MOI、0.1MOI和1MOI的接毒劑量時均可出現(xiàn)。接毒后不同時間收集上清并測定各組樣品毒價后提示MARC-5G2sc Fv細胞上清中病毒滴度顯著低于MARC-1B5scFv細胞和MARC-145細胞(P0.05),且在接毒后12h最為明顯(P0.001)。4.通過病毒吸附試驗和檢測I型干擾素表達初步探討胞內(nèi)表達5G2scFv下調(diào)病毒復制增殖的機制。熒光定量PCR和病毒滴度檢測結(jié)果顯示病毒吸附并無明顯改變(P0.05),提示胞內(nèi)表達5G2scFv不影響病毒吸附MARC-145細胞。I型干擾素轉(zhuǎn)錄和表達檢測結(jié)果顯示,接毒48h后,MARC-5G2scFv細胞中IFN-α的轉(zhuǎn)錄和表達較未感染的MARC-5G2scFv細胞及感染后MARC-1B5scFv和MARC-145細胞明顯升高(P0.05),而在MARC-1B5scFv和MARC-145細胞中無明顯變化(P0.05);但是三組細胞中IFN-β的轉(zhuǎn)錄和表達在感染前后均無明顯改變(P0.05)。以上結(jié)果提示胞內(nèi)表達5G2scFv抑制病毒感染可能與IFN-α表達上調(diào)有關(guān)。綜上所述,本研究通過構(gòu)建抗獨特型單鏈抗體并使其在MARC-145細胞中穩(wěn)定表達,對5G2scFv胞內(nèi)表達會否影響的PRRSV感染及其機制進行了一系列的研究。本試驗首次構(gòu)建穩(wěn)定表達抗獨特型單鏈抗體的MARC-145細胞系;證明其胞內(nèi)表達可抑制病毒復制增殖;該作用并非通過抑制病毒吸附實現(xiàn)而與IFN-α分泌增加相關(guān)。本研究拓展了抗獨特型單鏈抗體的應用,為尋找新的治療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.

【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：S858.28

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