本文關(guān)鍵詞：禽流感、新城疫和犬瘟熱病毒單克隆抗體篩選策略比較及應(yīng)用研究　出處：《東北農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 動物病毒 單克隆抗體 抗體應(yīng)用 治療性抗體 抗體測序

【摘要】：抗體是免疫球蛋白大蛋白家族成員,抗體蛋白結(jié)合特定表位,由淋巴細(xì)胞合成或由攻擊性(或非自體)有機(jī)體生成。單克隆抗體技術(shù)的發(fā)展和成熟為抗體應(yīng)用奠定了基礎(chǔ)。單克隆抗體具有針對單一抗原表位、能夠無限傳代、穩(wěn)定分泌表達(dá)等特點,在疾病診斷及治療等方面有著極為明顯的優(yōu)勢�？贵w除了疾病診斷及治療應(yīng)用外,還可作為基礎(chǔ)研究工具使用,根據(jù)其與抗原特異性結(jié)合特性,在研究中發(fā)揮作用。但是如何能獲得活性好、效價高的抗體一直是廣大免疫學(xué)及其相關(guān)學(xué)者研究的熱點和重點。本研究采用包括天然蛋白和重組蛋白在內(nèi)的不同免疫原進(jìn)行免疫,根據(jù)所篩選的抗體用途和特性,將ELISA、血凝抑制試驗及中和試驗等多種篩選方法相結(jié)合,以期對雜交瘤技術(shù)更高效的篩選到滿足需求的抗體進(jìn)行比較,為今后抗體制備策略提供重要參考依據(jù)。本研究通過對禽流感病毒(AIV)、新城疫病毒(NDV)和犬瘟熱病毒(CDV)3種動物重要疫病病毒的不同蛋白單克隆抗體的制備,從免疫原、篩選方法及制備過程等方面入手,共計獲得穩(wěn)定分泌的單抗21株。分別對制備的單抗特性、所針對的抗原表位進(jìn)行鑒定并進(jìn)行應(yīng)用研究探索。針對AIV共計制備獲得抗體7株。通過原核表達(dá),獲得了AIV-NS1蛋白,用重組蛋白免疫BALB/c小鼠。另外,經(jīng)SPF雞胚擴(kuò)增。AIV病毒擴(kuò)增后進(jìn)行蔗糖密度梯度離心純化,病毒純化后,于電鏡下觀察病毒粒子,然后同樣免疫BALB/c小鼠。應(yīng)用雜交瘤技術(shù)取免疫小鼠脾細(xì)胞與SP2/0細(xì)胞融合,AIV-NS1蛋白免疫小鼠用蛋白ELISA試驗篩選�？笰IV-NS1蛋白獲得抗體2株,分別命名為:D7和D9。用血凝抑制試驗結(jié)合AIV-ELISA篩選獲得抗AIV-HA單抗5株,分別命名為:D1C12、D3B2、D3B3、D3A11和D12B3。但所有5株均不具有中和活性。對AIV-NS1蛋白的兩株抗體D7及D9進(jìn)行抗原表位鑒定。分別用噬菌體展示肽技術(shù)對D7和D9各進(jìn)行了3輪生物淘選,并結(jié)合多肽芯片和截短表達(dá)技術(shù)鑒定出2株單抗的最短線性B細(xì)胞抗原表位。D7識別的抗原表位為29DAPF32;D9識別的抗原表位為182WNDNT186。經(jīng)SPF雞胚擴(kuò)增NDV,濃縮后免疫BALB/c小鼠。用NDV-HN原核表達(dá)蛋白ELISA試驗篩選到3株抗體,分別命名為:D5D2,D5G2和D6A2,該3株抗體均不具有血凝抑制活性及中和活性;用NDV全病毒ELISA方法篩選到抗體5株,依次命名為:D1B4,D1D9,D3F1,D4F8和D5E11,其中2株具有血凝抑制活性,但所有5株均不具有中和活性;用血凝抑制試驗篩選到抗體3株,分別命名為:D1H12,D5C5和D3C2,其中D5C5體外雞胚中和試驗結(jié)果顯示,其能夠?qū)姸綟48E9毒株、中等毒力Mukteswar毒株和弱毒La Sota毒株均具有中和作用,其中和效價均大于1:2048。經(jīng)截短表達(dá)鑒定發(fā)現(xiàn),D5C5所識別的抗原中和表位為353DEQDYQIR360。通過選取針對NDV活性高的2株單克隆抗體D1H12和D5C5,結(jié)合HRP-anti-NDV多抗血清,建立抗體夾心ELISA檢測試劑盒及膠體金檢測試紙條,結(jié)果能夠快速對新城疫進(jìn)行檢測,從而方便現(xiàn)地對于新城疫疫情的監(jiān)測。該檢測方法的特異性均良好,與雞痘病毒、傳染性支氣管炎病毒、傳染性喉氣管炎病毒、小鵝瘟病毒、雞毒支原體、雞傳染性法氏囊病病毒、禽流感病毒均未見反應(yīng)性。同時還發(fā)現(xiàn)ELISA與RT-PCR敏感性相同,均達(dá)到10-3尿囊液稀釋度;膠體金檢測試紙條能快速檢測NDV抗原,敏感性為10-2尿囊液稀釋度。在病毒蛋白與宿主蛋白相互作用的研究中,抗體作為工具也發(fā)揮了重要作用。本研究應(yīng)用針對NDV-HN蛋白的中和抗體與新城疫強毒F48E9感染的DF1細(xì)胞裂解后進(jìn)行免疫沉淀,獲得接毒與未接毒細(xì)胞的蛋白差異條帶,通過LTQ質(zhì)譜分析得出與NDV-HN蛋白有相互作用的兩個蛋白,即Caveolin和Apolipoprotein B。Caveolin是細(xì)胞表面微囊標(biāo)志蛋白,其與微囊形成和定位相關(guān),也參與胞吞和胞內(nèi)囊泡運輸、信號轉(zhuǎn)導(dǎo)、膽固醇穩(wěn)態(tài)和運輸。Apolipoprotein B在脂類和膽固醇的運輸和代謝過程中發(fā)揮重要作用。通過原核表達(dá)CDV-F蛋白免疫BALB/c小鼠。抗F蛋白獲得2株抗體,分別命名為:D1G8和D3F1,但與CDV反應(yīng)性弱,不具有中和活性。用Vero細(xì)胞擴(kuò)增CDV,通過蔗糖密度梯度離心方法對病毒進(jìn)行純化并通過電鏡觀察病毒粒子形態(tài)。免疫小鼠后用CDV全病毒ELISA方法結(jié)合中和試驗篩選到抗體1株,命名為DC2,該抗體具有中和活性,體外細(xì)胞中和試驗結(jié)果顯示,其中和效價為1:3200。結(jié)果表明,在篩選抗體之初,首先要明確所選抗體的主要用途,再結(jié)合抗原特性進(jìn)行設(shè)計,并結(jié)合多種篩選方法對抗體進(jìn)行篩選,對于獲得所需抗體的幾率會大幅提升。本研究進(jìn)而對通過雜交瘤技術(shù)制備的針對NDV、CDV的高中和活性抗體D5C5和DC2進(jìn)行動物體內(nèi)治療效果研究。選用Mukteswar中等毒力疫苗作為雛雞的感染毒株建立動物疾病模型,確定感染劑量為106EID50/只,之后對發(fā)病雛雞用中和抗體1mL/只進(jìn)行肌肉注射,結(jié)果發(fā)現(xiàn),患雛雞成活率由15%提高為60%,具有明確的體內(nèi)治療效果�？谷翢岵《局泻涂贵w靜脈推注CDV核酸陽性狐貍,結(jié)果發(fā)現(xiàn),注射抗體后患病狐貍不但臨診狀態(tài)明顯好轉(zhuǎn),而且其口腔、鼻拭子及血液中均未見檢出CDV核酸,表明該抗體具有體內(nèi)中和病毒的作用。對兩株具有中和活性的抗體的可變區(qū)測序,通過對5’RACE、高簡并引物及巢式混合引物PCR 3種方法的比較,最終確定巢式混合引物PCR具有較高的敏感性和特異性,可用于鼠源單抗的序列測定,結(jié)果獲得了D5C5和DC2的重鏈及輕鏈可變區(qū)序列。之后再根據(jù)可變區(qū)測序結(jié)果設(shè)計引物對其進(jìn)行擴(kuò)增,然后與鼠Ig Fc段連接,獲得了完整的鼠源抗體DNA。流式細(xì)胞術(shù)篩選分泌抗體的單個B淋巴細(xì)胞也是近幾年研究的熱點,本研究通過對經(jīng)濟(jì)動物狐貍的外周血淋巴細(xì)胞進(jìn)行分離后,再以Anti-CD19-FITC抗體作為探針對狐貍的B淋巴細(xì)胞進(jìn)行識別并分選,確定了該抗體可以識別狐貍的B淋巴細(xì)胞,為今后狐貍的相關(guān)疫病抗體篩選提供了技術(shù)支持。本研究從抗體制備、篩選方法入手,對獲得所需抗體的策略進(jìn)行較為全面的分析與闡述。從抗體應(yīng)用角度出發(fā),對本研究獲得的抗體進(jìn)行包括作為試驗工具、建立診斷方法以及體內(nèi)治療等方面的應(yīng)用研究。另外對常用的抗原表位鑒定方法進(jìn)行了比較分析,并選取3株抗體進(jìn)行了表位鑒定。對具有中和活性的2株抗體可變區(qū)進(jìn)行測序,并與鼠IgFc段連接獲得完整DNA。并且確定了可用于流式分選狐貍B淋巴細(xì)胞的抗體。對今后獸用抗體的制備、鑒定和應(yīng)用奠定了十分重要的技術(shù)基礎(chǔ)。
[Abstract]:Antibodies are protein protein family members of immunoglobulin binding protein, antibody specific epitopes by lymphocyte synthesis or by aggression (or non self) organisms to produce. The development of monoclonal antibody technology has laid the foundation for the application. And mature antibody monoclonal antibody with single epitope can infinitely, the characteristics of stable expression etc. that has a very obvious advantage in disease diagnosis and treatment. In addition to disease diagnosis and treatment of antibody application, can also be used as a basic research tool, according to the combination with antigen specific characteristics, play a role in the study. But how can obtain good activity, high antibody titer is always a hot key study on the immunology and related scholars. This research adopts different immune including natural protein and recombinant protein, primary immune antibody screening, according to the purposes and characteristics Of ELISA, the combination of hemagglutination inhibition and neutralization test and other screening methods, in order to screen hybridoma technology more efficient to meet the needs of the antibody were compared for antibody preparation strategies provide important reference basis. Through the study of avian influenza virus (AIV), Newcastle disease virus (NDV) and canine distemper virus (CDV) with different monoclonal antibody important disease virus 3 animal species were prepared from the immunogen, screening method and preparation process and other aspects, a total stable secretion of 21 mAbs. Of monoclonal antibody preparation, the epitopes were identified the application research for AIV. Total 7 strains. The prepared antibody obtained by prokaryotic expression, AIV-NS1 protein, BALB/c protein in mice immunized with recombinant SPF. In addition, the chicken embryo virus amplification amplification of.AIV after sucrose density gradient centrifugation, virus After purification, in the electron microscope observation of virus particles and then BALB/c mice were immunized by hybridoma technique. The immunized mouse spleen cells were fused with SP2/0 cells, AIV-NS1 mice immunized with protein ELISA screening test. The anti AIV-NS1 protein antibody obtained 2 strains, named D7 and D9. by hemagglutination inhibition test with AIV-ELISA screening anti AIV-HA monoclonal antibody in 5 strains, named D1C12, D3B2, D3B3, D3A11 and D12B3., but all of the 5 strains were not with neutralizing activity. The AIV-NS1 protein two monoclonal antibodies D7 and D9 epitope identification. By phage display peptide technology of D7 and D9 conducted 3 rounds of biopanning, and with the chip and the expression of truncated polypeptide identified 2 strains of monoclonal antibody the short-term B cell epitope.D7 recognition epitope 29DAPF32; D9 epitope recognition of 182WNDNT186. by SPF amplification of NDV concentrated chicken, immune BALB/c Rat. NDV-HN prokaryotic expression protein ELISA screening test to 3 strains of monoclonal antibodies, named D5D2, D5G2 and D6A2, the 3 antibodies have no hemagglutination inhibition activity and neutralizing activity; NDV virus ELISA method for screening antibodies to 5 strains, designated as D1B4, D1D9, D3F1, D4F8 and D5E11, of which 2 strains with hemagglutination inhibition activity, but not all of the 5 strains were with neutralizing activity; by hemagglutination inhibition test to the antibody screening 3 strains, named D1H12, D5C5 and D3C2, D5C5 in vitro embryo neutralization test results show that it can against virulent F48E9 strains in virulence of Mukteswar virulent and avirulent La strains of Sota have neutralizing effect, which was more than 1:2048. and the titer of the truncated expression identified D5C5 epitopes recognized by neutralizing epitope of 353DEQDYQIR360. by selecting for high activity NDV 2 monoclonal antibodies D1H12 and D5C5, combined with HRP-anti-NDV antiserum, The establishment of antibody sandwich ELISA detection kit and colloidal gold test strip, the result is capable of fast detection of Newcastle disease, which is convenient for monitoring the Newcastle disease epidemic site. The specificity of the method was good, with fowlpox virus, infectious bronchitis virus, infectious laryngotracheitis virus, gosling plague virus, Mycoplasma gallisepticum chicken, infectious bursal disease virus, avian influenza virus showed no reaction. It was also found that ELISA and RT-PCR have reached the same sensitivity, 10-3 allantoic fluid dilution; colloidal gold test strip can rapid detection of NDV antigen, the sensitivity of 10-2 allantoic fluid dilution. In the study of virus host interactions. As a tool, the antibody also plays an important role. This study applied for DF1 cell lysis protein NDV-HN neutralizing antibody and virulent Newcastle disease virus F48E9 infection after receiving immune precipitation Protein differences between virus and uninoculated cell bands by LTQ mass spectrometry analysis of two proteins interacting with NDV-HN protein, namely Caveolin and Apolipoprotein B.Caveolin is a cell surface protein markers of the microcapsules and microcapsules, the formation and orientation, is also involved in endocytosis and intracellular vesicular transport, signal transduction, and cholesterol homeostasis transport.Apolipoprotein B play an important role in the transport and metabolism of lipids and cholesterol. The expression of CDV-F protein in BALB/c mice immunized by prokaryotic protein. Anti F 2 monoclonal antibodies were obtained, named D1G8 and D3F1, but with CDV reaction is weak, does not have neutralizing activity. The amplification of CDV with Vero cells of the virus purified by sucrose density gradient centrifugation and observe the morphology of virus particles by electron microscopy. Mice immunized with CDV virus ELISA antibody screening method combined with neutralization test to 1 strains, named DC2, the antibody With neutralizing activity, in vitro neutralization test results showed that the titer of 1:3200., and results in antibody screening at the beginning, the main purpose should first clear the selected antibody, combined with the characteristics of antigen design, combined with a variety of screening methods for antibody screening, for the probability of gaining the desired antibodies will significantly enhance the. In order to study for the NDV prepared by hybridoma technology, CDV D5C5 and DC2 high school and the activity of antibody therapy were studied. The effects in vivo animal Mukteswar moderate vaccine strains were established as animal models of disease, determine the infection dose of 106EID50/, after the onset of chicks by neutralizing antibody 1mL/ only by intramuscular injection the results showed that, with the survival rate increased from 15% to 60%, with a clear treatment. In vivo against canine distemper virus neutralizing antibodies to intravenous injection of CDV nucleic acid positive Fox Beaver, found that after injection of antibody in the fox not only clinical status improved significantly, and the oral, nasal swabs and blood were not detected CDV nucleic acid, showed that the antibody with in vivo neutralization of. Sequences of variable region of antibodies with neutralizing activity against two strains, the 5 'RACE, relatively high degenerate primers and nested primers PCR 3 hybrid methods, and ultimately determine the mixed nested primer PCR has high sensitivity and specificity, can be used to determine the sequence of the murine antibody, we obtained sequence variable region of heavy chain and light chain of D5C5 and DC2. Then according to the results of sequencing primers were designed for the variable region was then connected with Ig in the Fc segment, the mouse antibody DNA. complete screening antibody secreting single B lymphocyte is the research hotspot in recent years by flow cytometry, through the study of peripheral blood of economic animal Fox Lymphocytes were isolated after using Anti-CD19-FITC antibody as a probe to the fox B lymphocytes were identified and sorted, identified the antibody can recognize fox B cells for future disease antibody screening fox provides technical support. This study from the antibody preparation, screening methods, to obtain the strategy of antibody a more comprehensive analysis and elaboration. From the Perspective of the application of antibodies, antibody obtained in this study were included as test tools, application of the establishment of diagnostic methods and in vivo treatment. In addition to the common epitope identification methods are analyzed, and selected 3 strains of antibody epitope identification by sequencing. With neutralizing activity of 2 strains of antibody variable region, and IgFc in connection to get the full DNA. and can be used to determine the flow sorting B lymphocyte anti Fox It has laid a very important technical basis for the preparation, identification and application of animal antibodies in the future.

【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S852.4

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