禽白血病病毒抗原/抗体ELISA检测方法的建立及准种分析
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摘要
禽白血病(Avian leukosis)是制约养禽业发展的主要禽类传染病,以垂直传播为主,免疫污染禽白血病病毒(Avian leukosis virus, ALV)的活疫苗也是引发该病的重要原因之一。目前主要通过定期对鸡群进行ALV血清学监测,采取淘汰净化的方法控制该病。我国开展ALV血清学监测通常使用美国IDEXX等公司的检测试剂盒,检测成本高、推广范围窄。因此,研究开发具有自主知识产权的ALV检测试剂盒,降低ALV检测成本,扩大鸡群检测范围,对我国防控本病有重要的实际应用意义。另外,ALV-J在临床上自报道以来一直处于不断的变异中,当前尚无确切证据证明ALV-J在我国鸡群中致病作用有多样性的机制,而病毒准种的多样性很有可能是ALV-J致病作用多样性的机制之一。因此,开展ALV准种研究对于从分子水平上解析ALV-J在我国不同鸡群中致病作用的多样性具有重要的理论意义。
本研究通过原核表达ALV p27蛋白,制备抗p27蛋白多克隆抗体,建立了ALV抗原ELISA检测方法和ALV抗体ELISA检测方法。试验结果表明,建立的两种检测方法与禽常见传染病病毒或其阳性血清、大肠杆菌阳性血清无交叉反应,具有较好的特异性；两种方法的批内、批间重复性试验变异系数均小于10%,具有良好的可重复性；ALV抗原ELISA检测方法与IDEXX公司的ALV抗原检测试剂盒敏感性相当,符合率达到98.9%；ALV抗体ELISA检测方法比IDEXX公司的ALVA/B和J亚群抗体检测试剂盒相对于间接免疫荧光试验(IFA)有更高的符合率。
利用本研究建立的抗原ELISA检测方法对918批不同疫苗进行了ALV检测,结果检测出63批外源性ALV污染疫苗。从2006年-2009年间污染的FPV. NDV和IBDV活疫苗中分别分离和鉴定到三株ALV,并对这三株ALV进行全基因组序列测定与分析。结果显示,三株ALV的gp85氨基酸序列与A亚群ALV参考毒株的gp85氨基酸序列同源性最高,为88.3%-97.7%,并且他们的全基因组整体同源性均较高；三株ALV与2009年分离自海兰褐祖代鸡的A亚群ALV中国分离株SDAU09C3同源性最高,显示它们很可能有共同的来源,也说明疫苗ALV污染是导致临床上鸡群发病的原因之一。
从山东省某地方品系鸡疑似白血病发病鸡群分离到自然感染的血管瘤型ALV-J,并采用本研究建立的ALV抗原／抗体ELISA检测方法进行了抗原和抗体验证,同时选取LY1201株对其env基因进行了比较分析。结果证明ALV-J单一病毒株中存在差异极大的不同准种,发现gp85在氨基酸位点210-219发生10个连续的氨基酸缺失的准种,该缺失在此前我国ALV-J野毒株中均未发现,该研究说明可通过gp85序列测定进行ALV准种分析研究。在此基础上,将ALV-J LY1201株在DF-1细胞上连传3代后,扩增其gp85基因并对其20个阳性克隆子进行测序和准种分析,结果发现在20个克隆中gp85缺失的突变准种已经完全消失,原来的优势准种比例由33.3%上升为85.0%成为绝对优势准种。这表明ALV-J在适应细胞复制过程中病毒的准种组成是不断变化的,且发生突变的准种只有其复制能力高于原优势准种才可能具备竞争性优势。
综上所述,本研究建立的ALV抗原/抗体ELISA检测方法,可用于ALV病毒检测和血清学监测,对于控制我国禽用病毒类活疫苗质量,加强禽白血病防控和净化具有重要的意义。目前本研究建立的ALV抗原ELISA检测方法已作为国家标准用于禽用病毒类活疫茁外源病毒检验。从禽用病毒类活疫苗中分离到污染的ALV,提示加强疫苗中ALV等外源病毒监测的重要性。同时,本研究建立的ALV-J准种分析方法,对于解析ALV-J在我国不同鸡群中致病作用的多样性提供了理论与技术支持。
Avian leukosis is one of the important poultry diseases which are restricting the development of the poultry industry. It spreads vertically mainly. Immunity of vaccines polluted by avian leukosis virus (ALV) is also one of the important routes of transmission of the disease. At present, mainly through regular ALV serology monitoring of chickens, elimination and purification method are adopted to control the disease. ALV serology monitoring in our country usually uses the United States IDEXX detection kits, which leads to high test cost and narrow promotion range. Therefore, the research and development with independent intellectual property rights of ALV detection kits, to reduce costs, expand detection range, has important practical significance in the disease prevention and control in China. In addition, ALV-J in clinic has been in constant variation since it was reported for the first time, and there is no any conclusive evidence to clarify the mechanism of the current of ALV-J in a variety of pathogenic role in chicken flocks in China, but virus quasispecies diversity is likely one of the mechanisms of ALV-J pathogenic role diversity. Therefore, carrying out the ALV quasispecies research, has important theoretical significance in analytical ALV-J at the molecular level in the diversity of different pathogenic role in chicken flocks in China
This research through the prokaryotic expression of ALV p27protein, preparation of p27protein polyclonal antibody, established the ALV antigen ELISA detection method and ALV antibody ELISA detection method. The two kinds of detection methods had no cross reaction with common avian infectious viruses or their positive serum and anti-e.coli positive serum, with good specificity; Coefficient of intra, inter variation of two methods were less than10%, with good repeatability; The sensitivity coincidence rate of ALV antigen ELISA detection method with IDEXX ALV antigen detection kit was98.9%; ALV antibody ELISA detection method had higher coincidence rate than IDEXX ALV A/B, J subgroup antibody detection kit relative to the indirect immunofluorescence test (IFA).
918different batches of vaccine were detected by ALV ELISA antigen detection method established in this study, the result showed that63batches of vaccine were polluted by exogenous ALV. From FPV, NDV and IBDV live vaccine polluted by ALV during2006year to2009year, three strains of ALV were isolated and identified respectively.Then the whole genome of the three strains of ALV were sequenced and analyzed. The results showed that the gp85gene homology of three strains and A subgroup of ALV reference strains was the highest, which was88.3%-97.7%, and their whole genome homology was also high; The highest homology of the three strains ALV and A subgroup of ALV, SDAU09C3strain, isolated from the brown highland progenitor generation chicken in China in2009, showed that they were likely to have a common source, also showed that the vaccine ALV pollution was one of the reasons leading to clinical disease of chickens.
From a local breed flock of Shandong province,9strains of natural infected hemangioma ALV-J were separated, and this research was verified by ALV antigen/antibody ELISA detection method established in this study. At the same time, LY1201strain was selected for the env gene comparative analysis. The results demonstrated the existence of a great different quasispecies in ALV-J single strains, which gp85occurres10consecutive amino acid deletions in210#-219#.The missing in the ALV-J gp85was not found in the wild strains in our country previous. The study showed that ALV quasispecies analysis can be carried out through ALV gp85sequence analysis. On this basis, the ALV-J LY1201strain passaged on DF-1cells after3generations, the gp85gene was amplified and sequenced. Quasispecies analysis20positive clones, found missing quasispecies in the mutant gp85had completely disappeared, the original advantage quasispecies ratio increased from33.3%to85%became the dominant quasispecies.This indicated that the ALV-J quasispecies were constantly changing in the cell replication process and the mutant quasispecies were difficult to become the dominant quasispecies only if they captured the high replication ability.
In summary, the ALV antigen/antibody ELISA detection method established in this study, can be used for ALV virus detection and serological surveillance, has important significance for controlling avian virus live vaccine quality and for strengthening the prevention and purification of avian leukosis. At present, the ALV antigen ELISA detection method established in this study has been used as the national standard in exogenous virus test of avian virus live vaccine. Isolation of the polluted ALV from avian virus live vaccine, are prompting the importance of strengthening ALV exogenous virus monitoring of the vaccine. At the same time, this study established a kind of quasispecies analysis method of ALV-J, providing a theoretical and technical support for parsing the diversity of different pathogenic role of ALV-J in chicken flocks in China.
本研究通过原核表达ALV p27蛋白,制备抗p27蛋白多克隆抗体,建立了ALV抗原ELISA检测方法和ALV抗体ELISA检测方法。试验结果表明,建立的两种检测方法与禽常见传染病病毒或其阳性血清、大肠杆菌阳性血清无交叉反应,具有较好的特异性；两种方法的批内、批间重复性试验变异系数均小于10%,具有良好的可重复性；ALV抗原ELISA检测方法与IDEXX公司的ALV抗原检测试剂盒敏感性相当,符合率达到98.9%；ALV抗体ELISA检测方法比IDEXX公司的ALVA/B和J亚群抗体检测试剂盒相对于间接免疫荧光试验(IFA)有更高的符合率。
利用本研究建立的抗原ELISA检测方法对918批不同疫苗进行了ALV检测,结果检测出63批外源性ALV污染疫苗。从2006年-2009年间污染的FPV. NDV和IBDV活疫苗中分别分离和鉴定到三株ALV,并对这三株ALV进行全基因组序列测定与分析。结果显示,三株ALV的gp85氨基酸序列与A亚群ALV参考毒株的gp85氨基酸序列同源性最高,为88.3%-97.7%,并且他们的全基因组整体同源性均较高；三株ALV与2009年分离自海兰褐祖代鸡的A亚群ALV中国分离株SDAU09C3同源性最高,显示它们很可能有共同的来源,也说明疫苗ALV污染是导致临床上鸡群发病的原因之一。
从山东省某地方品系鸡疑似白血病发病鸡群分离到自然感染的血管瘤型ALV-J,并采用本研究建立的ALV抗原／抗体ELISA检测方法进行了抗原和抗体验证,同时选取LY1201株对其env基因进行了比较分析。结果证明ALV-J单一病毒株中存在差异极大的不同准种,发现gp85在氨基酸位点210-219发生10个连续的氨基酸缺失的准种,该缺失在此前我国ALV-J野毒株中均未发现,该研究说明可通过gp85序列测定进行ALV准种分析研究。在此基础上,将ALV-J LY1201株在DF-1细胞上连传3代后,扩增其gp85基因并对其20个阳性克隆子进行测序和准种分析,结果发现在20个克隆中gp85缺失的突变准种已经完全消失,原来的优势准种比例由33.3%上升为85.0%成为绝对优势准种。这表明ALV-J在适应细胞复制过程中病毒的准种组成是不断变化的,且发生突变的准种只有其复制能力高于原优势准种才可能具备竞争性优势。
综上所述,本研究建立的ALV抗原/抗体ELISA检测方法,可用于ALV病毒检测和血清学监测,对于控制我国禽用病毒类活疫苗质量,加强禽白血病防控和净化具有重要的意义。目前本研究建立的ALV抗原ELISA检测方法已作为国家标准用于禽用病毒类活疫茁外源病毒检验。从禽用病毒类活疫苗中分离到污染的ALV,提示加强疫苗中ALV等外源病毒监测的重要性。同时,本研究建立的ALV-J准种分析方法,对于解析ALV-J在我国不同鸡群中致病作用的多样性提供了理论与技术支持。
Avian leukosis is one of the important poultry diseases which are restricting the development of the poultry industry. It spreads vertically mainly. Immunity of vaccines polluted by avian leukosis virus (ALV) is also one of the important routes of transmission of the disease. At present, mainly through regular ALV serology monitoring of chickens, elimination and purification method are adopted to control the disease. ALV serology monitoring in our country usually uses the United States IDEXX detection kits, which leads to high test cost and narrow promotion range. Therefore, the research and development with independent intellectual property rights of ALV detection kits, to reduce costs, expand detection range, has important practical significance in the disease prevention and control in China. In addition, ALV-J in clinic has been in constant variation since it was reported for the first time, and there is no any conclusive evidence to clarify the mechanism of the current of ALV-J in a variety of pathogenic role in chicken flocks in China, but virus quasispecies diversity is likely one of the mechanisms of ALV-J pathogenic role diversity. Therefore, carrying out the ALV quasispecies research, has important theoretical significance in analytical ALV-J at the molecular level in the diversity of different pathogenic role in chicken flocks in China
This research through the prokaryotic expression of ALV p27protein, preparation of p27protein polyclonal antibody, established the ALV antigen ELISA detection method and ALV antibody ELISA detection method. The two kinds of detection methods had no cross reaction with common avian infectious viruses or their positive serum and anti-e.coli positive serum, with good specificity; Coefficient of intra, inter variation of two methods were less than10%, with good repeatability; The sensitivity coincidence rate of ALV antigen ELISA detection method with IDEXX ALV antigen detection kit was98.9%; ALV antibody ELISA detection method had higher coincidence rate than IDEXX ALV A/B, J subgroup antibody detection kit relative to the indirect immunofluorescence test (IFA).
918different batches of vaccine were detected by ALV ELISA antigen detection method established in this study, the result showed that63batches of vaccine were polluted by exogenous ALV. From FPV, NDV and IBDV live vaccine polluted by ALV during2006year to2009year, three strains of ALV were isolated and identified respectively.Then the whole genome of the three strains of ALV were sequenced and analyzed. The results showed that the gp85gene homology of three strains and A subgroup of ALV reference strains was the highest, which was88.3%-97.7%, and their whole genome homology was also high; The highest homology of the three strains ALV and A subgroup of ALV, SDAU09C3strain, isolated from the brown highland progenitor generation chicken in China in2009, showed that they were likely to have a common source, also showed that the vaccine ALV pollution was one of the reasons leading to clinical disease of chickens.
From a local breed flock of Shandong province,9strains of natural infected hemangioma ALV-J were separated, and this research was verified by ALV antigen/antibody ELISA detection method established in this study. At the same time, LY1201strain was selected for the env gene comparative analysis. The results demonstrated the existence of a great different quasispecies in ALV-J single strains, which gp85occurres10consecutive amino acid deletions in210#-219#.The missing in the ALV-J gp85was not found in the wild strains in our country previous. The study showed that ALV quasispecies analysis can be carried out through ALV gp85sequence analysis. On this basis, the ALV-J LY1201strain passaged on DF-1cells after3generations, the gp85gene was amplified and sequenced. Quasispecies analysis20positive clones, found missing quasispecies in the mutant gp85had completely disappeared, the original advantage quasispecies ratio increased from33.3%to85%became the dominant quasispecies.This indicated that the ALV-J quasispecies were constantly changing in the cell replication process and the mutant quasispecies were difficult to become the dominant quasispecies only if they captured the high replication ability.
In summary, the ALV antigen/antibody ELISA detection method established in this study, can be used for ALV virus detection and serological surveillance, has important significance for controlling avian virus live vaccine quality and for strengthening the prevention and purification of avian leukosis. At present, the ALV antigen ELISA detection method established in this study has been used as the national standard in exogenous virus test of avian virus live vaccine. Isolation of the polluted ALV from avian virus live vaccine, are prompting the importance of strengthening ALV exogenous virus monitoring of the vaccine. At the same time, this study established a kind of quasispecies analysis method of ALV-J, providing a theoretical and technical support for parsing the diversity of different pathogenic role of ALV-J in chicken flocks in China.
引文
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