禽流感、新城疫和犬瘟热病毒单克隆抗体筛选策略比较及应用研究

发布时间：2018-01-18 16:06

本文关键词：禽流感、新城疫和犬瘟热病毒单克隆抗体筛选策略比较及应用研究　出处：《东北农业大学》2016年博士论文　论文类型：学位论文

【摘要】：抗体是免疫球蛋白大蛋白家族成员,抗体蛋白结合特定表位,由淋巴细胞合成或由攻击性(或非自体)有机体生成。单克隆抗体技术的发展和成熟为抗体应用奠定了基础。单克隆抗体具有针对单一抗原表位、能够无限传代、稳定分泌表达等特点,在疾病诊断及治疗等方面有着极为明显的优势。抗体除了疾病诊断及治疗应用外,还可作为基础研究工具使用,根据其与抗原特异性结合特性,在研究中发挥作用。但是如何能获得活性好、效价高的抗体一直是广大免疫学及其相关学者研究的热点和重点。本研究采用包括天然蛋白和重组蛋白在内的不同免疫原进行免疫,根据所筛选的抗体用途和特性,将ELISA、血凝抑制试验及中和试验等多种筛选方法相结合,以期对杂交瘤技术更高效的筛选到满足需求的抗体进行比较,为今后抗体制备策略提供重要参考依据。本研究通过对禽流感病毒(AIV)、新城疫病毒(NDV)和犬瘟热病毒(CDV)3种动物重要疫病病毒的不同蛋白单克隆抗体的制备,从免疫原、筛选方法及制备过程等方面入手,共计获得稳定分泌的单抗21株。分别对制备的单抗特性、所针对的抗原表位进行鉴定并进行应用研究探索。针对AIV共计制备获得抗体7株。通过原核表达,获得了AIV-NS1蛋白,用重组蛋白免疫BALB/c小鼠。另外,经SPF鸡胚扩增。AIV病毒扩增后进行蔗糖密度梯度离心纯化,病毒纯化后,于电镜下观察病毒粒子,然后同样免疫BALB/c小鼠。应用杂交瘤技术取免疫小鼠脾细胞与SP2/0细胞融合,AIV-NS1蛋白免疫小鼠用蛋白ELISA试验筛选。抗AIV-NS1蛋白获得抗体2株,分别命名为:D7和D9。用血凝抑制试验结合AIV-ELISA筛选获得抗AIV-HA单抗5株,分别命名为:D1C12、D3B2、D3B3、D3A11和D12B3。但所有5株均不具有中和活性。对AIV-NS1蛋白的两株抗体D7及D9进行抗原表位鉴定。分别用噬菌体展示肽技术对D7和D9各进行了3轮生物淘选,并结合多肽芯片和截短表达技术鉴定出2株单抗的最短线性B细胞抗原表位。D7识别的抗原表位为29DAPF32;D9识别的抗原表位为182WNDNT186。经SPF鸡胚扩增NDV,浓缩后免疫BALB/c小鼠。用NDV-HN原核表达蛋白ELISA试验筛选到3株抗体,分别命名为:D5D2,D5G2和D6A2,该3株抗体均不具有血凝抑制活性及中和活性;用NDV全病毒ELISA方法筛选到抗体5株,依次命名为: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;用血凝抑制试验筛选到抗体3株,分别命名为:D1H12,D5C5和D3C2,其中D5C5体外鸡胚中和试验结果显示,其能够对强毒F48E9毒株、中等毒力Mukteswar毒株和弱毒La Sota毒株均具有中和作用,其中和效价均大于1:2048。经截短表达鉴定发现,D5C5所识别的抗原中和表位为353DEQDYQIR360。通过选取针对NDV活性高的2株单克隆抗体D1H12和D5C5,结合HRP-anti-NDV多抗血清,建立抗体夹心ELISA检测试剂盒及胶体金检测试纸条,结果能够快速对新城疫进行检测,从而方便现地对于新城疫疫情的监测。该检测方法的特异性均良好,与鸡痘病毒、传染性支气管炎病毒、传染性喉气管炎病毒、小鹅瘟病毒、鸡毒支原体、鸡传染性法氏囊病病毒、禽流感病毒均未见反应性。同时还发现ELISA与RT-PCR敏感性相同,均达到10-3尿囊液稀释度;胶体金检测试纸条能快速检测NDV抗原,敏感性为10-2尿囊液稀释度。在病毒蛋白与宿主蛋白相互作用的研究中,抗体作为工具也发挥了重要作用。本研究应用针对NDV-HN蛋白的中和抗体与新城疫强毒F48E9感染的DF1细胞裂解后进行免疫沉淀,获得接毒与未接毒细胞的蛋白差异条带,通过LTQ质谱分析得出与NDV-HN蛋白有相互作用的两个蛋白,即Caveolin和Apolipoprotein B。Caveolin是细胞表面微囊标志蛋白,其与微囊形成和定位相关,也参与胞吞和胞内囊泡运输、信号转导、胆固醇稳态和运输。Apolipoprotein B在脂类和胆固醇的运输和代谢过程中发挥重要作用。通过原核表达CDV-F蛋白免疫BALB/c小鼠。抗F蛋白获得2株抗体,分别命名为:D1G8和D3F1,但与CDV反应性弱,不具有中和活性。用Vero细胞扩增CDV,通过蔗糖密度梯度离心方法对病毒进行纯化并通过电镜观察病毒粒子形态。免疫小鼠后用CDV全病毒ELISA方法结合中和试验筛选到抗体1株,命名为DC2,该抗体具有中和活性,体外细胞中和试验结果显示,其中和效价为1:3200。结果表明,在筛选抗体之初,首先要明确所选抗体的主要用途,再结合抗原特性进行设计,并结合多种筛选方法对抗体进行筛选,对于获得所需抗体的几率会大幅提升。本研究进而对通过杂交瘤技术制备的针对NDV、CDV的高中和活性抗体D5C5和DC2进行动物体内治疗效果研究。选用Mukteswar中等毒力疫苗作为雏鸡的感染毒株建立动物疾病模型,确定感染剂量为106EID50/只,之后对发病雏鸡用中和抗体1mL/只进行肌肉注射,结果发现,患雏鸡成活率由15%提高为60%,具有明确的体内治疗效果。抗犬瘟热病毒中和抗体静脉推注CDV核酸阳性狐狸,结果发现,注射抗体后患病狐狸不但临诊状态明显好转,而且其口腔、鼻拭子及血液中均未见检出CDV核酸,表明该抗体具有体内中和病毒的作用。对两株具有中和活性的抗体的可变区测序,通过对5’RACE、高简并引物及巢式混合引物PCR 3种方法的比较,最终确定巢式混合引物PCR具有较高的敏感性和特异性,可用于鼠源单抗的序列测定,结果获得了D5C5和DC2的重链及轻链可变区序列。之后再根据可变区测序结果设计引物对其进行扩增,然后与鼠Ig Fc段连接,获得了完整的鼠源抗体DNA。流式细胞术筛选分泌抗体的单个B淋巴细胞也是近几年研究的热点,本研究通过对经济动物狐狸的外周血淋巴细胞进行分离后,再以Anti-CD19-FITC抗体作为探针对狐狸的B淋巴细胞进行识别并分选,确定了该抗体可以识别狐狸的B淋巴细胞,为今后狐狸的相关疫病抗体筛选提供了技术支持。本研究从抗体制备、筛选方法入手,对获得所需抗体的策略进行较为全面的分析与阐述。从抗体应用角度出发,对本研究获得的抗体进行包括作为试验工具、建立诊断方法以及体内治疗等方面的应用研究。另外对常用的抗原表位鉴定方法进行了比较分析,并选取3株抗体进行了表位鉴定。对具有中和活性的2株抗体可变区进行测序,并与鼠IgFc段连接获得完整DNA。并且确定了可用于流式分选狐狸B淋巴细胞的抗体。对今后兽用抗体的制备、鉴定和应用奠定了十分重要的技术基础。
[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.

【学位授予单位】：东北农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.4

【参考文献】