【摘要】：牛傳染性鼻氣管炎病毒（Infectious bovine rhinotracheitis virus，IBRV）可引起牛發(fā)生急性、熱性、接觸性傳染病--牛傳染性鼻氣管炎（Infectious bovine rhinotracheitis，IBR）。主要表現(xiàn)為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，能誘導(dǎo)機(jī)體產(chǎn)生保護(hù)性抗體的抗原，因此gD可作為鑒別診斷IBR的首選特異性抗原。本試驗以IBRV的gD基因為研究對象，通過原核表達(dá)蛋白后，用其建立檢測IBRV抗體的間接ELISA診斷方法，并進(jìn)行初步應(yīng)用，目的是期望在臨床實踐中能夠快速檢測和診斷牛傳染性鼻氣管炎病，為今后該病的檢驗檢疫和凈化提供技術(shù)支持。本研究主要進(jìn)行了如下兩個方面的研究工作： 一、IBRV gD基因的原核表達(dá)根據(jù)Genbank上發(fā)表的gD基因序列，設(shè)計合成3對特異性引物，利用PCR擴(kuò)增出gD基因3段表位即gD-A、gD-B、gD-C，并構(gòu)建原核表達(dá)重組質(zhì)粒pET-28a-gD。將其轉(zhuǎn)入BL21（DE3）表達(dá)菌中，利用IPTG誘導(dǎo)表達(dá)。經(jīng)酶切鑒定及基因測序均證明原核表達(dá)載體構(gòu)建成功。SDS-PAGE表明，gD融合蛋白主要以包涵體形式在大腸埃希菌中高效表達(dá)，成功獲得了大小約為48ku的融合蛋白，與預(yù)期的蛋白分子量一致。純化后的gD重組蛋白濃度為0.128mg/mL，免疫印跡的結(jié)果顯示純化后的gD重組蛋白能與IBR標(biāo)準(zhǔn)陽性血清發(fā)生特異性反應(yīng)，說明其免疫原性良好。 二、IBRV抗體間接ELISA方法建立與初步應(yīng)用以純化的IBRV gD蛋白為包被抗原，建立檢測IBRV抗體的間接ELISA方法。通過對各種反應(yīng)條件的優(yōu)化，確定最佳包被抗原濃度為6.4μg/mL，包被時間為4℃過夜，血清稀釋度為1:50，血清反應(yīng)條件為37℃1h，封閉液為5%脫脂乳，HRP-兔抗牛IgG的最佳工作濃度為1:10000，，最佳顯色時間為37℃15min，經(jīng)統(tǒng)計學(xué)分析后確定的陰陽性臨界值為0.219。結(jié)果顯示，利用本研究所建立的ELISA方法不但特異性強(qiáng)，而且重復(fù)性穩(wěn)定。應(yīng)用本試驗所建立檢測方法和IDEXX試劑盒，分別對442份來自黑龍江省部分地區(qū)未經(jīng)免疫的牛血清樣本進(jìn)行檢測，結(jié)果顯示在442份血清樣本中102份樣本IBR抗體呈陽性，血清陽性率為23.1%，與IDEXX試劑盒達(dá)到90.2%的符合率。臨床檢測結(jié)果顯示，所建立的檢測方法具有特異、敏感和穩(wěn)定性好的優(yōu)點，應(yīng)用前景廣闊。
[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

本文編號：2360359