中华鳖虹彩病毒单克隆抗体的制备、特性分析及应用研究
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摘要
以纯化的中华鳖虹彩病毒为抗原免疫Balb/C小鼠,经细胞杂交融合、筛选,制备出分泌抗中华鳖虹彩病毒特异性单克隆抗体的细胞系,分析了中华鳖虹彩病毒单克隆抗体的免疫生物学特性,建立了能够用于检测中华鳖虹彩病毒的ELISA、IFA方法,应用中华鳖虹彩病毒单克隆抗体对蛙虹彩病毒属STIV、RGV、FJIV三株病毒的抗原性进行了初步的比较分析。获得如下结果:
1、中华鳖虹彩病毒的提取与纯化:对中华鳖虹彩病毒的三种不同的纯化方法进行比较分析,结果表明两次冻融后差速离心的方法纯化病毒纯度差,回收率较低;磁力搅拌加上超声处理后差速离心可以提高病毒粒子的回收率,但容易造成病毒结构破坏;匀浆、磁力搅拌,差速离心结合蔗糖密度梯度离心方法可获得高纯度的病毒样品,电镜观察和SDS-PAGE分析进一步证实最后一种方法提纯的病毒纯度高、结构完好,可用于免疫Balb/C鼠。
2、中华鳖虹彩病毒单克隆抗体的制备:以纯化的中华鳖虹彩病毒抗原免疫Balb/C小鼠,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行杂交融合,经过初筛、复筛和三次有限稀释法克隆,获得8个能稳定分泌抗中华鳖虹彩病毒特异性单克隆抗体杂交瘤细胞株,分别命名为Mab2A4、Mab8E1、Mab1D3、Mab2H1、Mab3A1、Mab485、Mab5E1和Mab6F2。
3、中华鳖虹彩病毒单克隆抗体免疫生物学特性分析:单克隆抗体亚级份分析结果表明,Mab2A4属IgA,Mab8E1是IgG2a,其他的6株单抗1D3、2H1、3A1、485、5E1和6F2的亚级份均为IgG1。ELISA分析表明,8株单抗均能特异性地识别中华鳖虹彩病毒抗原,与EPC、Co、FHM等宿主细胞抗原不产生任何交叉反应,腹水抗体的ELISA效价在10~(-4)～10~(-6)之间。IFA分析表明,8株单抗中仅Mab5E1没有免疫荧光反应特性,其余7株单抗均能对染毒病灶产生特异性的荧光染色。中和试验结果证实8株单抗均没有中和病毒的特性。Western-blot结果显示,Mab1D3和Mab2A4分别识别分子量为84kDa和35kDa中华鳖虹彩病毒结构蛋白,Mab3A1能够同时识别分子量分别为14kDa、15kDa和16kDa的三条多肽,其余单抗不具备Western-blot反应特性。这些结果提示上述单抗对中华鳖虹彩病毒抗原高度特异、灵敏,可用于中华鳖虹彩病毒的检测和结构蛋白分析。
4、单克隆抗体在虹彩病毒研究中的应用:应用中华鳖虹彩病毒单克隆抗体对蛙病毒属的STIV、RGV、FJIV三株病毒进行交叉反应,结果表明8株单抗与三种不同来源的蛙病毒抗原均有ELISA反应特性,除了Mab5E1外,其余单抗与三种不同来源的蛙病毒抗原均有IFA反应特性。结果表明蛙病毒属的STIV、RGV、FJIV三株病毒抗原性高度一致,可能提示蛙病毒属属特异性抗原位点高度保守。
虹彩病毒结构复杂,揭示虹彩病毒的致病和免疫机理难度大。中华鳖虹彩病毒单克隆抗体的成功制备为分析蛙病毒属病毒的结构蛋白与功能,病毒复制、装配,以及侵染宿主和康复过程病毒粒子的分布与消长提供了十分有用的工具,并奠定重要的技术基础。
In this study, the purified antigen of soft-shelled turtle iridovirus (STIV) was used to immunize the Balb/C mice. After fusion and selection, the hybridoma lines secreting monoclonal antibodies against STIV were produced. And the immune biological characteristics were analyzed by the indirect enzyme linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA) and western blot. The antigen-detection ELISA and IFA systems were developed. Moreover, these monoclonal antibodies were preliminarily used to compare and analyze the antigen of three strains STIV, RGV and FJIV, which belongs to Ranavirus genus. The results were as following.
1. Extraction and purification of soft-shelled turtle iridovirus:
Three different purification methods were used and compared. The results indicated that a number of cell-associated viral particles were discarded with cell debris during centrifugation if freezing-thawing was used only before differential purification. In order to maximize the yield, the method of the magnetic force stir and sonication was used to purify the viral suspensions, but it will easily scathe and destroy the full virion. A abundant highly purified virion could be isolated using differential and sucrose gradient centrifugation after the treatment with the combination of freezing-thawing、the magnetic force stirring, and homogenization. And the virus particles were observed by transmission electron microscope after differential and sucrose grading, and the virions were intact and in shape. The purified virus proteins were analyzed by SDS-PAGE and confirmed to be the better immunogen to immunize Balb/C mice.
2. Production of monoclonal antibody to the soft-shelled turtle iridovirus:
The Balb/C mice were immunized with purified soft-shelled turtle iridovirus antigen. And then the mouse spleen cells were fused with SP_2/O myeloma cells using standard procedures. After primary and secondary screening, the positive hybridomas were selected and cloned using three cycles of limiting dilution. Eight stable monoclonal hybridomas designated 1D3, 2A4, 2H1, 3A1, 4B5, 5E1, 6F2 and 8E1 were produced.
3. Characterization of monoclonal antibody to the soft-shelled turtle iridovirus:
The Mabs obtained were three kinds of isotype. Mab 2A4 was subclass IgA, Mab8El was subclass IgG2a, and the other Mabs 1D3, 2H1, 3A1, 4B5, 5E1 and 6F2 were subclass IgG1. ELISA assays showed that eight Mabs could specifically recognize the antigen of STIV, and had no cross reaction with the cell lines EPC, Co, FHM. The ELISA titers of ascites were between 10~(-4) and 10~(-6). Immunofluorescent studies showed that all Mabs (except for Mab5El) had fluorescence characteristics, and the specific fluorescence signals appeared virus assembly sites in the cytoplasm of STIV-infected (EPC) cells. None of the Mabs possessed the ability to neutralize SGIV in vitro cell cultures. Western blot analysis demonstrated that Mabs 1D3 and 2A4 reacted specifically to a single linear protein with an approximately molecular weight of 50kDa and 25kDa respectively, Mab3A1 reacted with three STIV proteins at molecular mass of about 14-16kDa. The identity of the target antigen of the other five Mabs could not be determined by western blot analysis. These Mabs against STIV might be highly specific and sensitive, and they might be used to detect soft-shelled turtle iridovirus and analyze its structure proteins.
4. Application of monoclonal antibody in iridovirus:
The eight monoclonal antibodies against STIV were used to analyze three different virus strains STIV, FJIV and RGV. ELISA assay revealed that all of the monoclonal antibodies produced specifically positive reactions with the three strains. Detected by IFA, the seven Mabs (except for Mab5E1) showed specific fluorescence signals. The results showed that the antigenicity of STIV, RGV and FJIV is significantly similar, which suggest that Ranavirus sp. had highly conservative antigenic epitopes.
The iridovirus structure is very complicated, so it was difficult to elucidate the pathogenesis and immune mechanisms. The successfully production of STIV-specific Mabs will provide an important basis to analyze the structure and function proteins of Ranavirus sp., virus replication and assembly. The Mabs may prove to be a very useful tool in the study of the distribution and fluctuation of virions after invasion and rehabilitation processes.
1、中华鳖虹彩病毒的提取与纯化:对中华鳖虹彩病毒的三种不同的纯化方法进行比较分析,结果表明两次冻融后差速离心的方法纯化病毒纯度差,回收率较低;磁力搅拌加上超声处理后差速离心可以提高病毒粒子的回收率,但容易造成病毒结构破坏;匀浆、磁力搅拌,差速离心结合蔗糖密度梯度离心方法可获得高纯度的病毒样品,电镜观察和SDS-PAGE分析进一步证实最后一种方法提纯的病毒纯度高、结构完好,可用于免疫Balb/C鼠。
2、中华鳖虹彩病毒单克隆抗体的制备:以纯化的中华鳖虹彩病毒抗原免疫Balb/C小鼠,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行杂交融合,经过初筛、复筛和三次有限稀释法克隆,获得8个能稳定分泌抗中华鳖虹彩病毒特异性单克隆抗体杂交瘤细胞株,分别命名为Mab2A4、Mab8E1、Mab1D3、Mab2H1、Mab3A1、Mab485、Mab5E1和Mab6F2。
3、中华鳖虹彩病毒单克隆抗体免疫生物学特性分析:单克隆抗体亚级份分析结果表明,Mab2A4属IgA,Mab8E1是IgG2a,其他的6株单抗1D3、2H1、3A1、485、5E1和6F2的亚级份均为IgG1。ELISA分析表明,8株单抗均能特异性地识别中华鳖虹彩病毒抗原,与EPC、Co、FHM等宿主细胞抗原不产生任何交叉反应,腹水抗体的ELISA效价在10~(-4)～10~(-6)之间。IFA分析表明,8株单抗中仅Mab5E1没有免疫荧光反应特性,其余7株单抗均能对染毒病灶产生特异性的荧光染色。中和试验结果证实8株单抗均没有中和病毒的特性。Western-blot结果显示,Mab1D3和Mab2A4分别识别分子量为84kDa和35kDa中华鳖虹彩病毒结构蛋白,Mab3A1能够同时识别分子量分别为14kDa、15kDa和16kDa的三条多肽,其余单抗不具备Western-blot反应特性。这些结果提示上述单抗对中华鳖虹彩病毒抗原高度特异、灵敏,可用于中华鳖虹彩病毒的检测和结构蛋白分析。
4、单克隆抗体在虹彩病毒研究中的应用:应用中华鳖虹彩病毒单克隆抗体对蛙病毒属的STIV、RGV、FJIV三株病毒进行交叉反应,结果表明8株单抗与三种不同来源的蛙病毒抗原均有ELISA反应特性,除了Mab5E1外,其余单抗与三种不同来源的蛙病毒抗原均有IFA反应特性。结果表明蛙病毒属的STIV、RGV、FJIV三株病毒抗原性高度一致,可能提示蛙病毒属属特异性抗原位点高度保守。
虹彩病毒结构复杂,揭示虹彩病毒的致病和免疫机理难度大。中华鳖虹彩病毒单克隆抗体的成功制备为分析蛙病毒属病毒的结构蛋白与功能,病毒复制、装配,以及侵染宿主和康复过程病毒粒子的分布与消长提供了十分有用的工具,并奠定重要的技术基础。
In this study, the purified antigen of soft-shelled turtle iridovirus (STIV) was used to immunize the Balb/C mice. After fusion and selection, the hybridoma lines secreting monoclonal antibodies against STIV were produced. And the immune biological characteristics were analyzed by the indirect enzyme linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA) and western blot. The antigen-detection ELISA and IFA systems were developed. Moreover, these monoclonal antibodies were preliminarily used to compare and analyze the antigen of three strains STIV, RGV and FJIV, which belongs to Ranavirus genus. The results were as following.
1. Extraction and purification of soft-shelled turtle iridovirus:
Three different purification methods were used and compared. The results indicated that a number of cell-associated viral particles were discarded with cell debris during centrifugation if freezing-thawing was used only before differential purification. In order to maximize the yield, the method of the magnetic force stir and sonication was used to purify the viral suspensions, but it will easily scathe and destroy the full virion. A abundant highly purified virion could be isolated using differential and sucrose gradient centrifugation after the treatment with the combination of freezing-thawing、the magnetic force stirring, and homogenization. And the virus particles were observed by transmission electron microscope after differential and sucrose grading, and the virions were intact and in shape. The purified virus proteins were analyzed by SDS-PAGE and confirmed to be the better immunogen to immunize Balb/C mice.
2. Production of monoclonal antibody to the soft-shelled turtle iridovirus:
The Balb/C mice were immunized with purified soft-shelled turtle iridovirus antigen. And then the mouse spleen cells were fused with SP_2/O myeloma cells using standard procedures. After primary and secondary screening, the positive hybridomas were selected and cloned using three cycles of limiting dilution. Eight stable monoclonal hybridomas designated 1D3, 2A4, 2H1, 3A1, 4B5, 5E1, 6F2 and 8E1 were produced.
3. Characterization of monoclonal antibody to the soft-shelled turtle iridovirus:
The Mabs obtained were three kinds of isotype. Mab 2A4 was subclass IgA, Mab8El was subclass IgG2a, and the other Mabs 1D3, 2H1, 3A1, 4B5, 5E1 and 6F2 were subclass IgG1. ELISA assays showed that eight Mabs could specifically recognize the antigen of STIV, and had no cross reaction with the cell lines EPC, Co, FHM. The ELISA titers of ascites were between 10~(-4) and 10~(-6). Immunofluorescent studies showed that all Mabs (except for Mab5El) had fluorescence characteristics, and the specific fluorescence signals appeared virus assembly sites in the cytoplasm of STIV-infected (EPC) cells. None of the Mabs possessed the ability to neutralize SGIV in vitro cell cultures. Western blot analysis demonstrated that Mabs 1D3 and 2A4 reacted specifically to a single linear protein with an approximately molecular weight of 50kDa and 25kDa respectively, Mab3A1 reacted with three STIV proteins at molecular mass of about 14-16kDa. The identity of the target antigen of the other five Mabs could not be determined by western blot analysis. These Mabs against STIV might be highly specific and sensitive, and they might be used to detect soft-shelled turtle iridovirus and analyze its structure proteins.
4. Application of monoclonal antibody in iridovirus:
The eight monoclonal antibodies against STIV were used to analyze three different virus strains STIV, FJIV and RGV. ELISA assay revealed that all of the monoclonal antibodies produced specifically positive reactions with the three strains. Detected by IFA, the seven Mabs (except for Mab5E1) showed specific fluorescence signals. The results showed that the antigenicity of STIV, RGV and FJIV is significantly similar, which suggest that Ranavirus sp. had highly conservative antigenic epitopes.
The iridovirus structure is very complicated, so it was difficult to elucidate the pathogenesis and immune mechanisms. The successfully production of STIV-specific Mabs will provide an important basis to analyze the structure and function proteins of Ranavirus sp., virus replication and assembly. The Mabs may prove to be a very useful tool in the study of the distribution and fluctuation of virions after invasion and rehabilitation processes.
引文
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