应用重组ALV-J gp85干酪乳杆菌多克隆抗体检测J亚型禽白血病间接ELISA方法的建立
发布时间：2018-07-17 07:28
【摘要】：J亚群禽白血病病毒(Avian Leukosis Virus Subgroup J,ALV-J)自1988年从肉鸡中分离出来,在世界范围内广泛流传。以垂直和水平两种传播方式传播,诱发肿瘤、引起生长缓慢、消瘦、死亡为特征。在种鸡上还可降低产蛋率、受精率、孵化率。同时患病鸡骨髓、胸腺、法氏囊发育受阻导致机体发生免疫抑制,不能产生足够的抗体应对疾病,导致疫苗免疫失效,易于患病,给养禽业带来巨大的经济损失。近年来,该病在我国养鸡业中呈现流行趋势,优质地方土鸡、三黄鸡、商品蛋鸡中都有发病情况报道。目前临床上尚无有效的药物和疫苗能够用于此病的治疗,鉴于上述原因只有通过淘汰ALV-J阳性鸡,建立无ALV-J病原的净化鸡群,才是解决当前问题的唯一手段。因此,建立双抗夹心ELISA检测出血清中的病毒,及时淘汰带毒鸡对防制该病流行、开展种群净化具有重要意义。根据大多数病毒和细菌都是通过消化道和呼吸道的黏膜侵入动物机体的特性,利用重组活载体疫苗,采用口服免疫的方式把表达的抗原呈递到黏膜组织,以此来诱导全身的免疫应答来抵抗外界的细菌、病毒的入侵。乳酸杆菌作为一种自然界分布极为普遍,并广泛分布于动物机体内的具有益生保健作用的菌类,近年来乳酸菌口服疫苗已成为研制的热点。重组的乳酸菌通过天然的形式口服介导黏膜免疫,方法简便、安全,不仅在局部黏膜产生免疫应答,还可介导全身性的免疫应答。当把重组的含外源基因表达载体的乳酸杆菌作为活载体菌株时,能将乳酸杆菌的生物学特性和外源性抗原基因的免疫原性相结合,同时具有表达的外源蛋白不易受内毒素的影响、无包涵体并易于表达到细胞外的特点。重组的菌株易于保存扩繁,也符合新型基因工程口服疫苗的特点。本研究使用的方法是通过重组GP85囊膜糖蛋白的干酪乳杆菌口服免疫小白鼠,诱导小白鼠产生特异性抗体,免疫接种后每周检测抗体水平。6W后经琼脂双扩散法、Western-blot、直接ELISA检测合格的鼠抗ALV-J多抗,经提存后对此抗体进行酶标,作为酶标抗体。并利用本实验室保存的鼠单抗作为捕获抗体包被ELISA板,鼠多抗作为酶标二抗,建立双抗夹心ELISA方法。并对双抗夹心ELISA方法工作条件进行优化,来检测经DF1传代培养后禽血清中ALV-J。结果表明,封闭液浓度为5%的脱脂奶粉;单抗包被量为1μg/mL;酶标二抗的稀释度为1:1600;二抗作用时间为1h;底物显色时间为15min。经特异性试验、重复性试验、灵敏性实验和临界值确定结果显示,该方法具有良好的特性,与新城疫病毒、马立克病毒、传染性法氏囊病毒、传染性支气管炎病毒均无交叉反应,并通过对临床大量样品的检测,得到较为科学的阳性率,可以为临床检测ALV-J提供依据。
[Abstract]:Avian Leukosis virus (Avian Leukosis virus subgroup ALV-J) was isolated from broilers in 1988 and has been widely spread all over the world. It is characterized by vertical and horizontal transmission, inducing tumor, causing slow growth, wasting and death. The laying rate, fertilization rate and hatching rate can also be reduced on the broiler. At the same time, the development of bone marrow, thymus and bursa of diseased chicken lead to immunosuppression, which can not produce enough antibody to deal with the disease, which lead to vaccine failure, easy to fall ill, and bring huge economic loss to poultry industry. In recent years, the disease is popular in the chicken industry of our country. There are reports of the disease in the high quality local chicken, Sanhuang chicken and commercial laying hens. At present, there are no effective drugs and vaccines for the treatment of this disease. Only by eliminating ALV-J positive chickens and establishing purified chickens without ALV-J pathogen is the only way to solve the current problem. Therefore, it is of great significance to establish a double antibody sandwich Elisa to detect the virus in serum and to eliminate the virus in time to control the epidemic of the disease and to carry out population purification. Based on the fact that most viruses and bacteria invade the animal body through the mucous membrane of the digestive tract and the respiratory tract, the expressed antigen is presented to the mucosal tissue by oral immunization using the recombinant live vector vaccine. In order to induce a systemic immune response to resist the invasion of external bacteria, viruses. Lactobacillus is a kind of probiotic bacteria which is widely distributed in nature and widely distributed in animal organism. In recent years oral lactic acid bacteria vaccine has become a hot spot. Recombinant lactobacillus mediated mucosal immunity by oral administration in natural form. The method is simple and safe. It not only produces immune response in local mucosa, but also mediates systemic immune response. When the recombinant Lactobacillus containing exogenous gene expression vector was used as a living vector, the biological characteristics of Lactobacillus could be combined with the immunogenicity of exogenous antigen gene. At the same time, the extraneous proteins were easy to be affected by endotoxin, without inclusion bodies and easy to express out of cells. The recombinant strain is easy to preserve and propagate, and conforms to the characteristics of new genetic engineering oral vaccine. The method used in this study was to induce specific antibody production in mice by oral immunization with Lactobacillus casei, a recombinant GP85 envelope glycoprotein. The antibody level was detected weekly after immunization. The antibody level was detected by Western-blot method with Agar double diffusion method. The qualified mouse anti-ALV-J polyantibody was detected by direct Elisa. The antibody was labeled as enzyme labeled antibody after storage. The double antibody sandwich Elisa method was established by using the mouse McAb preserved in our laboratory as the capture antibody coating Elisa plate and the mouse polyantibody as the enzyme labeled second antibody. The working conditions of double antibody sandwich Elisa were optimized to detect ALV-J in avian serum after subculture of DF1. The results showed that the concentration of the sealant was 5%, the coating amount of the McAb was 1 渭 g / mL, the dilution of the second antibody was 1: 1600, the time of the second antibody was 1 h and the color reaction time of the substrate was 15 min. The results of specificity test, repeatability test, sensitivity test and critical value determination show that this method has good characteristics with Newcastle disease virus, Marek virus, infectious bursal virus, Infectious bronchitis virus has no cross reaction, and a scientific positive rate can be obtained by detecting a large number of clinical samples, which can provide the basis for clinical detection of ALV-J.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3
本文编号：2129600
[Abstract]:Avian Leukosis virus (Avian Leukosis virus subgroup ALV-J) was isolated from broilers in 1988 and has been widely spread all over the world. It is characterized by vertical and horizontal transmission, inducing tumor, causing slow growth, wasting and death. The laying rate, fertilization rate and hatching rate can also be reduced on the broiler. At the same time, the development of bone marrow, thymus and bursa of diseased chicken lead to immunosuppression, which can not produce enough antibody to deal with the disease, which lead to vaccine failure, easy to fall ill, and bring huge economic loss to poultry industry. In recent years, the disease is popular in the chicken industry of our country. There are reports of the disease in the high quality local chicken, Sanhuang chicken and commercial laying hens. At present, there are no effective drugs and vaccines for the treatment of this disease. Only by eliminating ALV-J positive chickens and establishing purified chickens without ALV-J pathogen is the only way to solve the current problem. Therefore, it is of great significance to establish a double antibody sandwich Elisa to detect the virus in serum and to eliminate the virus in time to control the epidemic of the disease and to carry out population purification. Based on the fact that most viruses and bacteria invade the animal body through the mucous membrane of the digestive tract and the respiratory tract, the expressed antigen is presented to the mucosal tissue by oral immunization using the recombinant live vector vaccine. In order to induce a systemic immune response to resist the invasion of external bacteria, viruses. Lactobacillus is a kind of probiotic bacteria which is widely distributed in nature and widely distributed in animal organism. In recent years oral lactic acid bacteria vaccine has become a hot spot. Recombinant lactobacillus mediated mucosal immunity by oral administration in natural form. The method is simple and safe. It not only produces immune response in local mucosa, but also mediates systemic immune response. When the recombinant Lactobacillus containing exogenous gene expression vector was used as a living vector, the biological characteristics of Lactobacillus could be combined with the immunogenicity of exogenous antigen gene. At the same time, the extraneous proteins were easy to be affected by endotoxin, without inclusion bodies and easy to express out of cells. The recombinant strain is easy to preserve and propagate, and conforms to the characteristics of new genetic engineering oral vaccine. The method used in this study was to induce specific antibody production in mice by oral immunization with Lactobacillus casei, a recombinant GP85 envelope glycoprotein. The antibody level was detected weekly after immunization. The antibody level was detected by Western-blot method with Agar double diffusion method. The qualified mouse anti-ALV-J polyantibody was detected by direct Elisa. The antibody was labeled as enzyme labeled antibody after storage. The double antibody sandwich Elisa method was established by using the mouse McAb preserved in our laboratory as the capture antibody coating Elisa plate and the mouse polyantibody as the enzyme labeled second antibody. The working conditions of double antibody sandwich Elisa were optimized to detect ALV-J in avian serum after subculture of DF1. The results showed that the concentration of the sealant was 5%, the coating amount of the McAb was 1 渭 g / mL, the dilution of the second antibody was 1: 1600, the time of the second antibody was 1 h and the color reaction time of the substrate was 15 min. The results of specificity test, repeatability test, sensitivity test and critical value determination show that this method has good characteristics with Newcastle disease virus, Marek virus, infectious bursal virus, Infectious bronchitis virus has no cross reaction, and a scientific positive rate can be obtained by detecting a large number of clinical samples, which can provide the basis for clinical detection of ALV-J.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3
【引证文献】
相关会议论文 前1条
1 潘伟;高玉龙;秦立廷;祁小乐;高宏雷;孙芬芬;王永强;邓晓芸;王笑梅;;蛋鸡J亚群禽白血病病毒HLJ09MDJ-1株的分离鉴定[A];中国畜牧兽医学会兽医公共卫生学分会第二次学术研讨会论文集[C];2010年
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