牛传染性鼻气管炎病毒gD基因的表达及ELISA诊断方法的建立

发布时间：2018-11-27 10:10

【摘要】：牛传染性鼻气管炎病毒（Infectious bovine rhinotracheitis virus，IBRV）可引起牛发生急性、热性、接触性传染病--牛传染性鼻气管炎（Infectious bovine rhinotracheitis，IBR）。主要表现为呼吸道和生殖道感染。IBRV主要的基因包括gB、gC、gD、gE和TK基因，其中糖蛋白gD位于IBRV囊膜表面，能诱导机体产生保护性抗体的抗原，因此gD可作为鉴别诊断IBR的首选特异性抗原。本试验以IBRV的gD基因为研究对象，通过原核表达蛋白后，用其建立检测IBRV抗体的间接ELISA诊断方法，并进行初步应用，目的是期望在临床实践中能够快速检测和诊断牛传染性鼻气管炎病，为今后该病的检验检疫和净化提供技术支持。本研究主要进行了如下两个方面的研究工作： 一、IBRV gD基因的原核表达根据Genbank上发表的gD基因序列，设计合成3对特异性引物，利用PCR扩增出gD基因3段表位即gD-A、gD-B、gD-C，并构建原核表达重组质粒pET-28a-gD。将其转入BL21（DE3）表达菌中，利用IPTG诱导表达。经酶切鉴定及基因测序均证明原核表达载体构建成功。SDS-PAGE表明，gD融合蛋白主要以包涵体形式在大肠埃希菌中高效表达，成功获得了大小约为48ku的融合蛋白，与预期的蛋白分子量一致。纯化后的gD重组蛋白浓度为0.128mg/mL，免疫印迹的结果显示纯化后的gD重组蛋白能与IBR标准阳性血清发生特异性反应，说明其免疫原性良好。 二、IBRV抗体间接ELISA方法建立与初步应用以纯化的IBRV gD蛋白为包被抗原，建立检测IBRV抗体的间接ELISA方法。通过对各种反应条件的优化，确定最佳包被抗原浓度为6.4μg/mL，包被时间为4℃过夜，血清稀释度为1:50，血清反应条件为37℃1h，封闭液为5%脱脂乳，HRP-兔抗牛IgG的最佳工作浓度为1:10000，，最佳显色时间为37℃15min，经统计学分析后确定的阴阳性临界值为0.219。结果显示，利用本研究所建立的ELISA方法不但特异性强，而且重复性稳定。应用本试验所建立检测方法和IDEXX试剂盒，分别对442份来自黑龙江省部分地区未经免疫的牛血清样本进行检测，结果显示在442份血清样本中102份样本IBR抗体呈阳性，血清阳性率为23.1%，与IDEXX试剂盒达到90.2%的符合率。临床检测结果显示，所建立的检测方法具有特异、敏感和稳定性好的优点，应用前景广阔。
[Abstract]:Bovine infectious rhinotracheitis virus (Infectious bovine rhinotracheitis virus,IBRV) can cause bovine infectious rhinotracheitis (Infectious bovine rhinotracheitis,IBR). The main genes of IBRV include gB,gC,gD,gE and TK genes, in which glycoprotein gD is located on the surface of IBRV capsule and can induce the body to produce antigens of protective antibodies. Therefore, gD can be used as the first choice specific antigen for differential diagnosis of IBR. In this study, the gD gene of IBRV was used as the research object. After prokaryotic expression of the protein, the indirect ELISA diagnostic method for detecting IBRV antibody was established, and its preliminary application was carried out. The aim of this study is to rapidly detect and diagnose bovine infectious rhinotracheitis in clinical practice, and to provide technical support for inspection, quarantine and purification of bovine infectious rhinotracheitis in the future. The main work of this study is as follows: 1. The prokaryotic expression of, IBRV gD gene is based on the gD gene sequence published on Genbank. Three pairs of specific primers were designed and synthesized, and three epitopes of gD gene, gD-A, were amplified by PCR. Construction of prokaryotic expression plasmid pET-28a-gD. by gD-B,gD-C, It was transferred into BL21 (DE3) expression bacteria and induced by IPTG. SDS-PAGE showed that the gD fusion protein was highly expressed in Escherichia coli in the form of inclusion body, and the fusion protein of about 48ku was obtained successfully. The molecular weight of the protein is in accordance with the expected molecular weight. The concentration of purified gD recombinant protein was 0.128 mg / mL. The results of Western blot showed that the purified gD recombinant protein could react specifically with IBR standard positive serum, indicating that the immunogenicity of the purified recombinant gD protein was good. Secondly, the indirect ELISA method for the detection of IBRV antibody was established and applied. The purified IBRV gD protein was used as the coating antigen to establish an indirect ELISA method for the detection of IBRV antibody. By optimizing the reaction conditions, the best coating antigen concentration was 6.4 渭 g / mL, the coating time was 4 鈩

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