【作者】 定明；

【导师】 刘正飞；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2010， 硕士

【摘要】 牛传染性鼻气管炎(Infectious Bovine Rhinotracheitis, IBR),是由牛疱疹病毒1型(BHV-1)引起的一种牛的急性、热性、接触性传染病,主要引起呼吸道和生殖道疾病,如传染性鼻气管炎、眼结膜炎、高热、传染性脓疱性外阴道炎、流产等。自被发现以来,该病就在世界范围内流行,给养牛业造成巨大的经济损失。IBR被国际兽疫局(0IE)列为B类疾病,该病的根治方法是阳性牛检出与捕杀,而防制的主要措施是接种疫苗,主要采用基因工程疫苗的方法,国外也已相继开发出了牛疱疹病毒1型TK、gE基因缺失重组疫苗。BHV-1是有囊膜的双股DNA病毒,其基因组G+C含量为72%,大小约为138kb,由一个UL区(106kb)和一个US区(10kb)及US区两侧的重复序列(IRs和TRs,各1lkb,序列相同但方向相反)组成。BHV-1基因组编码70个左右基因,其中有10种为糖蛋白。其中gE糖蛋白是病毒囊膜上的主要糖蛋白。它以细胞特异的方式影响病毒从被感染细胞中释放,同时gE基因的缺失也影响到病毒在细胞间的传播。而胸腺激酶(TK)基因是牛疱疹病毒1型增殖的非必需基因,也是主要毒力基因之一,对病毒在神经组织的持续感染具有非常重要的作用。本研究构建了牛疱疹病毒1型TK／gE双基因缺失突变株,并做了动物安全性和保护力试验,为研究缺失标志活病毒疫苗及研制表达外源基因的BHV-1重组病毒活载体疫苗奠定基础。主要工作包括：1.牛传染性鼻气管炎TK~-／gE~-基因双缺失突变株的构建1)牛传染性鼻气管炎TK-/EGFP+突变株的构建本实验以牛疱疹病毒1型全序列(基因登录号为AJ004801)为基础,提取病毒全基因组。在此基础上设计引物,用PCR技术扩增TK基因上游和下游约1.0 kb和1.1kb的片段作为同源重组臂,分别克隆于载体pBluescriptⅡSK(+)中。用相应的限制性内切酶将下游同源臂回收,定向插入到上游的克隆载体中,构建中间转移载体,并命名为pZF08-21。然后插入带有完整启动子的EGFP报告基因,构建成重组转移质粒,并命名为pZF07-16。以磷酸钙转染法将经线性化的pZF07-16、质粒pBICP0与BHV-1基因组DNA在牛肾细胞(MDBK)上共转染,成功地进行了同源重组,7轮筛选和纯化获得了BHV-1 TK-/EGFP+突变株。2)牛传染性鼻气管炎TK~-突变株的构建提取BHV-1 TK-/EGFP+突变株基因组,再次以磷酸钙转染法将经线性化的pZF08-21、质粒pBICP0与基因组DNA在MDBK上共转染,同源重组去掉EGFP荧光标签,经5轮纯化成功的获得了BHV-1 TK-突变株。3)牛传染性鼻气管炎TK-/gE-/EGFP+双缺失突变株的构建方法同1,用PCR扩增gE基因上游和下游约1.0kb和1.1kb的片段,分别克隆于载体pcDNA3.1(+)myc-His B中。通过限制性酶切将上游同源臂回收,插入到下游的克隆载体中,构建中间转移载体,并命名为pZF09-08。然后插入EGFP报告基因,构建成重组转移质粒,并命名为pZF09-15。以磷酸钙转染法将经线性化的pZF09-15、质粒pBICP0与BHV-1 TK"缺失突变株基因组DNA在MDBK细胞上共转染,成功地进行同源重组,经过7轮筛选和纯化获得了BHV-1 TK-/gE-/EGFP+突变株。2.牛传染性鼻气管炎TK-/gE-/EGFP+双缺失突变株的安全性和保护力研究1)牛传染性鼻气管炎TK-/gE-/EGFP+双缺失突变株对牛的安全性研究安全性研究包括三部分：接种试验、同居试验、再激活试验。接种试验和同居试验：将试验组按剂量分为105,106,107三栏,滴鼻免疫4X105、4X106、4X107PFU的双缺失突变株,另设空白对照组。另在各栏中同时喂养未接种的牛作为同居对照。测量试验牛的体温,观察精神状态和采食情况,采集检测样品。再激活试验：接种免疫28天后,连续3天以15mg／头的剂量肌肉注射地塞米松,以激活处于潜伏感染状态的病毒。试验证实了牛传染性鼻气管炎TK-/gE-/EGFP+双缺失突变株具有很高的安全性。2)牛传染性鼻气管炎TK-/gE-/EGFP+双缺失突变株对牛的保护力研究两月龄牛分别用4X105、4X106、4X107PFU的双缺失突变株免疫4周后,以4X107PFU BHV-1强毒攻击。同时设免疫DMEM的空白对照组。分别于攻毒后1d、2d、3d、5d、7d、14d、21d和28d测量体温、采集检测样品。试验结果表明：牛传染性鼻气管炎TK-/gE-/EGFP+双缺失突变株对牛具有很强的保护力。总之,本研究表明,BHV-1 TK-/gE-可以作为候选疫苗毒株,为我国牛传染性鼻气管炎根除计划提供工具。更多还原

【Abstract】 Infectious bovine rhinotracheitis (IBR), caused by bovine herpesvirus type 1 (BHV-1), is known as an acute, pyrexic and contagious disease. It’s a major pathogen of cattle causing respiratory and genital tract infections such as bovine rhinotracheitis, conjunctivitis, fever, infectious pustular vulvovaginitis, and abortion. Since BHV-1 was discovered, IBR has occurred all over the world, and causes great economic loss to the cattle and milk industry. IBR is ranked in list B diseases by OIE, and the main measure for eradication is to slaughter the infected cattle. In addition to this, Immunization is an effective way to control it. Many live BHV-1 vaccines has been developed such as BHV-1 TK null, BHV-1 gE null and so on.The genome of BHV-1 is a double-strand DNA of about 138 kb, with the G+C content of 72%. It contains two unique areas called the long unique (UL) and the short unique(US), and two reverse repeat units, e.t. IR and TR, flanking the US area. BHV-1 encodes 10 glycoproteins, including glycoprotein E (gE). gE is a non-essential gene for virus growth, but it influences cell-to-cell transmission in MDBK cell monolayer. On the other hand, thymidine kinase (TK) gene, another main virulence determing gene, is also non-essential for virus proliferation. It plays a very important role in the latency infection in the nervous system.The objective of this study is to construct a bovine herpersvirus type 1 live modified vaccine strain with TK gene and gE gene deletion, providing a platform for a live viral vector expressing exogenous genes. Safety and efficacy of the both TK gene and gE gene deleted vaccine strain was carried out. The research results are summarized as follows:1. Construction of BHV-1 TK-/gE-/EGFP+ double gene-deleted mutants1). Construction of BHV-1 TK-/EGFP+ gene-deleted mutantTwo pairs of primers of TK gene was synthesized based on the genomic DNA sequence of BHV-1 (GenBank Accession No. AJ004801). The upstream (1.0kb) and downstream (1.1kb) of TK gene homologous arms were amplified by PCR, sequenced, and were directly cloned into pBluescriptⅡSK(+) vector one by one by restriction enzymes. Then EGFP expression cassette was inserted in between the two homologous arms. The recombinant plasmid was linearized and co-transfect into MDBK cells together with pBICPO and BHV-1 wild-type genomic DNA. By green fluorescence screening and plaque purification, the recombinant BHV-1 TK-/EGFP+ was generated.2). Construction of BHV-1 TK- gene-deleted mutantThe pZF08-21 was linearized and co-transfected into MDBK cells together with pBICPO and BHV-1 TK-/EGFP+ genomic DNA. Through five rounds of flurescence selection and plaque purification, EGFP expression cassette was successfully removed, and BHV-1 TK- gene-deleted virus was generated.3). Construction of BHV-1 TK-/gE-/EGFP+ double gene-deleted mutantTwo pairs of primers of gE gene was synthesized based on the genomic DNA sequence of BHV-1 (GenBank accession No. AJ004801). The upstream (1.1kb) and downstream (1.0kb) of gE gene homologous arms were amplified by PCR, sequenced, and were directly cloned into pcDNA3.1(+) myc-His B vector one by one by restriction enzymes. The EGFP expression cassette was inserted in between the two homologous arms. The transfer plasmid was linearized and co-transfected into MDBK cells together with pBICPO and BHV-1 TK- genomic DNA. Through nine rounds of flurescence selection and plaque purification, BHV-1 TK-/gE-/EGFP+ double gene-deleted mutant was generated.2. The safety and efficacy of BHV-1 TK-/gE-/EGFP+ double gene-deleted vaccine strain in cattle1)The safety of BHV-1 TK-/gE-/EGFP+ double gene-deleted mutantThe safety experiment was divided into three parts.They were inoculation experiment, cohabitation experiment and reactivation experiment. Three different immune dose of vaccine strain (4×105PFU,4×106PFU,4×107PFU) were infected intranasally in 10 cattle. In addition, unvaccinated cattle was designated as cohabitation test in the inoculation group. Body temperature was mesured daily, serum samples and swabs were collected. Then, dexamethasone were injected for three days to reactivate the latent virus.The safe experiment shows that the TK-/gE-/EGFP+ gene-deleted mutant is quite safe for cattle.2)The efficacy of BHV-1 TK-/gE-/EGFP+ double gene-deleted mutantVaccination group and negative control group were classified in this experiment. Three different immune dose of vaccine strain (4×105PFU,4×106PFU,4×107PFU) were used to infect 9 cattle of two month old in the vaccination group, while DMEM were used in the negative control group (6 cattle). Four weeks post inoculation, both vaccination group and the control group were challenged with 4×107PFU BHV-1 wild-type virus intranasally. Body temperature was mesured daily, serum samples and swabs were collected on 1d,2d, 3d,5d,7d,14d,21d and 28d post challenging. The efficacy experiment shows that the BHV-1 TK-/gE- double gene-deleted virus could provide full immunoprotectivity to cattle.更多还原