鸡传染性法氏囊病毒单克隆抗体识别表位的鉴定
发布时间：2018-09-06 12:25
【摘要】：传染性法氏囊病毒(IBDV)会导致鸡发生严重的免疫抑制疾病,从而引起巨大的经济损失。自然条件下,IBDV广泛存在于周围环境中并且可以通过呼吸进行传播。IBDV感染的靶器官主要是法氏囊,会引起法氏囊肿大和出血等症状,使机体的免疫力下降,对其它病原因子的抵抗力下降,从而继发感染多种疾病。IBDV抗原结合表位是病毒分子感染宿主细胞的主要部位,也是药物治疗的靶序列。如果能找到正确的病毒表位,在IBDV感染宿主之前,采取相应措施切断病毒的感染途径,就能起到很好的保护作用。 本实验室根据已经报道的VP2蛋白的三维晶体结构,合成了VP2蛋白的五段序列,发现其中一条多肽P22可能是IBDV的抗原表位。方法本次实验将P22多肽进行了原核表达,发现其主要以包涵体形式存在。用镍柱纯化带His标签的P22多肽,纯化后的P22多肽放入透析袋中透析复性。SDS-PAGE检验多肽的纯化程度,Western blot检测P22多肽的特性。用IBD抗原快速检测试纸条验证Western blot结果。15日龄的SPF鸡颈部皮下接种P22多肽,二免后取P22抗血清。琼脂扩散实验和中和效价测定P22抗血清的特异性和保护细胞能力。结果SDS-PAGE结果表明获得了比较纯的P22多肽。Western blot结果表明P22多肽可以和IBDV单抗上清、IBDV标准阳性血清起特异反应,和IBDV阴性血清不反应。P22多肽可以与IBD快速检测试纸条发生特异反应,试纸条的控制线和检测线均显示红色条带。琼脂扩散实验显示P22抗血清和IBDV强毒株可以形成明显的免疫沉淀线,IBDV强毒株和IBDV单抗上清之间也有清晰的免疫沉淀线。随机选择3份P22抗血清,在Vero细胞上检测中和效价,结果分别是1：640,1：650和1：620。结论P22是IBDV VP2蛋白上的抗原决定簇,P22是VP2的线性B细胞抗原表位。
[Abstract]:Infectious bursal virus (IBDV) can lead to severe immunosuppressive disease in chickens and cause huge economic losses. Under natural conditions, IBDV is widely present in the surrounding environment and can be transmitted through respiration. The target organ of IBDV infection is mainly the bursa of Fabricius, which can cause symptoms such as bursal enlargement and bleeding and decrease the immunity of the body. IBDV antigen-binding epitopes are the main sites of virus molecules infecting host cells and are also the target sequences of drug therapy because of the decrease of resistance to other pathogenic factors and the secondary infection of many diseases with IBDV antigen binding epitopes. If we can find the correct epitope of the virus and take appropriate measures to cut off the infection pathway of the virus before IBDV infects the host, it can play a good protective role. According to the three-dimensional crystal structure of VP2 protein, we synthesized the five-segment sequence of VP2 protein, and found that one of the polypeptides P22 may be the epitope of IBDV. Methods the prokaryotic expression of P22 polypeptide was found to be mainly in the form of inclusion body. P22 polypeptide with His label was purified by nickel column. The purified P22 peptide was dialyzed in dialysis bag. SDS-PAGE was used to detect the purification degree of P22 polypeptide. Western blot was used to detect the characteristics of P22 polypeptide. P22 peptide was subcutaneously inoculated on the neck of 15-day-old SPF chicken with IBD antigen test strip. P22 antiserum was obtained after two immunizations. Agar diffusion assay and neutralization titer were used to determine the specificity and cell protection ability of P22 antiserum. Results the results of SDS-PAGE showed that P22 polypeptide. Western blot showed that P22 peptide could react specifically with IBDV McAb supernatant IBDV standard positive serum and IBDV negative serum. P22 polypeptide could react with IBD rapid test strip. Test strip control line and test line are shown in red strip. Agar diffusion test showed that P22 antiserum and IBDV virulent strain could form a clear immunoprecipitation line between IBDV virulent strain and IBDV monoclonal antibody supernatant. Three samples of P22 antiserum were randomly selected and neutralized in Vero cells. The results were 1: 64040: 650 and 1: 620, respectively. Conclusion P22 is an antigenic determinant of IBDV VP2 protein and a linear B cell epitope of VP2.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
本文编号：2226346
[Abstract]:Infectious bursal virus (IBDV) can lead to severe immunosuppressive disease in chickens and cause huge economic losses. Under natural conditions, IBDV is widely present in the surrounding environment and can be transmitted through respiration. The target organ of IBDV infection is mainly the bursa of Fabricius, which can cause symptoms such as bursal enlargement and bleeding and decrease the immunity of the body. IBDV antigen-binding epitopes are the main sites of virus molecules infecting host cells and are also the target sequences of drug therapy because of the decrease of resistance to other pathogenic factors and the secondary infection of many diseases with IBDV antigen binding epitopes. If we can find the correct epitope of the virus and take appropriate measures to cut off the infection pathway of the virus before IBDV infects the host, it can play a good protective role. According to the three-dimensional crystal structure of VP2 protein, we synthesized the five-segment sequence of VP2 protein, and found that one of the polypeptides P22 may be the epitope of IBDV. Methods the prokaryotic expression of P22 polypeptide was found to be mainly in the form of inclusion body. P22 polypeptide with His label was purified by nickel column. The purified P22 peptide was dialyzed in dialysis bag. SDS-PAGE was used to detect the purification degree of P22 polypeptide. Western blot was used to detect the characteristics of P22 polypeptide. P22 peptide was subcutaneously inoculated on the neck of 15-day-old SPF chicken with IBD antigen test strip. P22 antiserum was obtained after two immunizations. Agar diffusion assay and neutralization titer were used to determine the specificity and cell protection ability of P22 antiserum. Results the results of SDS-PAGE showed that P22 polypeptide. Western blot showed that P22 peptide could react specifically with IBDV McAb supernatant IBDV standard positive serum and IBDV negative serum. P22 polypeptide could react with IBD rapid test strip. Test strip control line and test line are shown in red strip. Agar diffusion test showed that P22 antiserum and IBDV virulent strain could form a clear immunoprecipitation line between IBDV virulent strain and IBDV monoclonal antibody supernatant. Three samples of P22 antiserum were randomly selected and neutralized in Vero cells. The results were 1: 64040: 650 and 1: 620, respectively. Conclusion P22 is an antigenic determinant of IBDV VP2 protein and a linear B cell epitope of VP2.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
【参考文献】
相关期刊论文 前2条
1 杨艳艳,张改平,郭军庆,陈凤民,袁麦顺;高亲和力抗IBDV单克隆抗体的产生与免疫学鉴定[J];中国免疫学杂志;2000年07期
2 张改平,李学伍,杨艳艳,邓瑞广,李青梅;鸡传染性法氏囊病快速诊断试纸条的研制及特性测定[J];畜牧兽医学报;2004年01期
，本文编号：2226346
本文链接：https://www.wllwen.com/xiyixuelunwen/2226346.html
最近更新
教材专著
