【作者】 李河林；

【导师】 王晶钰；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2010， 硕士

【摘要】 传染性牛鼻气管炎病( Infectious bovine rhinotracheitis, IBR)是由牛疱疹病毒1型(BHV 1)引起的急性、热性、接触性传染病,主要牛的引起严重呼吸道和生殖道感染,给全球养牛业造成巨大的经济损失,国际兽医局(OIE)将本病列为B类疾病,也是我国进境动物必检疾病之一。BHV-1基因编码11种糖蛋白,主要糖蛋白gB和gD高度保守,是病毒的吸附、穿入和在细胞之间扩散病毒必需的,均能诱导产生较高水平的体液和细胞免疫,尤其gD能够引起比gB更强而持久的细胞免疫,而且gD基因可引起外周血单核细胞和牛淋巴细胞的凋亡,可促成感染牛免疫抑制。基于此,本实验进行了如下三个方面的研究工作。1牛传染性鼻气管炎病毒XA株的分离与鉴定用MDBK细胞从陕西发病奶牛鼻拭子中分离牛传染性鼻气管炎病毒;使用IBR标准阳性血清对该病毒分离物进行中和试验;应用primer 5.0软件设计,根据Genbank公布IBRV的基因组序列,利用gB保守序列设计1对引物,对分离病毒进行PCR扩增,并测序。结果表明:分离病毒在MDBK细胞上产生明显的特征性细胞病变(CPE):细胞圆缩,聚集成葡萄串样群落,在单层细胞上形成空洞,有几个细胞核的巨大细胞;用标准抗IBRV特异性抗血清能完全中和分离株;用IBRV gB特异性引物进行PCR,可扩增出1182bp的特异性条带,目的序列与发表的IBRV的基因组序列一致。证明该分离株为牛传染性鼻气管炎病毒,命名为IBRV XA株。2牛传染性鼻气管炎病毒PCR检测方法的建立及应用参考GenBank中收录的IBRV gB基因序列,应用Primer5.0软件设计了一对引物,扩增目的片段为1182bp。同时对IBRV PCR检测方法进行了特异性、敏感性试验,并应用其对临床疑似发病牛血清及国内部分厂家生产的细胞培养用牛血清进行了检测。结果表明,该方法不仅具有较高的特异性,而且可检测出103.4TCID50/0.1mL的病毒量,病毒DNA最低检出量相当于2.16ng/20ul。98份样品中共检出12份阳性,阳性率为12.2%,其中陕西省不同地区牛场血清样品阳性率为12.8%(11/86),国内部分厂家生产的血清阳性率为8.3%(1/12)。说明该方法可用于IBRV的临床检测及流行病学监测。3牛传染性鼻气管炎病毒gD蛋白的原核表达参考GenBank中收录的IBRV gD基因序列,应用Primer5.0软件设计引物,用PCR方法成功扩增IBRV gD全基因,将其定向克隆到原核表达载体pET-32a(+)中,构建了gD基因的原核重组表达质粒pET-32a-gD。原核重组表达质粒pET-32a-gD转化大肠杆菌BL21(DE3),在异丙基硫代半乳糖苷(IPTG)诱导下表达gD蛋白,SDS-PAGE和Western blot分析表达产物表明,表达产物具有与IBRV相同的抗原性。更多还原

【Abstract】 Infectious bovine rhinotracheitis(IBR),which was caused by the Bovine herpersvirus-1(BHV-1),was acute, pyrexic and contagious, caused severe respiratory and genital infections in cattle and incurred great economic loss to the global cattle husbandry. IBR was listed as the B disease by OIE, and was the main object quarantined of the animals imported to China.BHV 1 genome encodes eleven glycoproteins. among them, glycoprotein B and glycoprotein D were the most highly conserved herpesvirus glycoprotein, have been shown to be essential for are involved in crucial steps during virus infection, in particular attachment to and penetration into target cells and direct cell-to-cell spread. They can induce high titer cellular and humoral immune respone, especially gD, it induces a more consistent and stronger cellular immune response than gB. And gD triggers apoptosis in peripheral blood mononuclear cells and bovine lymphoma cells, which could contribute to immunodepression in infected animals. Based on them, threes aspects of research works have been carried out in this paper.1 Isolation and identification of infectious bovine rhinotracheitis virusA infectious bovine rhinotracheitis(IBR) virus was isolated from a sick cow nasal swabs with (MDCK) cells, and the neutralizing antibody against the isolated virus were detected by the standard IBRV specific serum. A specific pair of primers were designed according to the gene sequence of IBRV gB gene publicated in GeneBank. The gB gene of the IBRV strain was amplified by PCR method, and the sequences was analyzed.The results showed that obvious characteristics of the cytopathic effect(CPE) generated in MDCK, such as grape-like clusters of rounded cells gathered around a hole in the monolayer; sometimes giant cells with several nuclei may be observed;The virus can be neutralized completely by the standard IBRV specific serum. Specific band can be amplicified by PCR with IBRV gB specific primers and the sequencing result showed that the sequence of the isolated virus was coincided with the published IBRV gB sequences. The results indicated that the isolate was IBRV, and be named IBRV XA strain.2 Establishment and Application of PCR for Detection Methods of IBRVA specific pair of primers were designed according to the gene sequence of infectious bovine rhinotracheitis virus (IBRV)gB gene publicated in GeneBank. The gB gene of the IBRV strain was amplified by PCR method, and the motive fragment of amplification is 1182bp. The PCR detective method was verified by specificity and sensibility tests. Meanwhile, Using the method to detect the serum samples which come from clinic bovine and part of manufacturers in China. The results showed that the method not only has high specificity, but also can examine virus with 103.4TCID50/0.1mL , The minimum detectable amount of virus DNA was 2.16ng/20ul。There were 12 positive samples in 98 sera, positive rate was 12.2%,among them clinic bovine and part of manufacturers in China were 12.8% and 8.3% Respectively. And it was considered to be the foundation for detecting and epidemiologic survey of IBRV.3 Prokaryotic expression of glycoprotein D of IBRVA specific pair of primers were designed according to the gene sequence of infectious bovine rhinotracheitis virus (IBRV)gB gene publicated in GeneBank. gD gene was amplified by PCR and cloned directly into the pET-32a(+),the recombinant prokaryotic expression plasmid pET-32a-gD was successfully constructed. pET-32a-gD was transformed into E.coli BL21(DE3), and induced with IPTG, the expressed products(gD proteins) had antigenicity same as IBRV which were analyzed with SDS-PAGE and Western blotting.更多还原