【作者】 郑海洲；

【导师】 赵宝华；

【作者基本信息】 河北师范大学 ， 细胞生物学， 2004， 硕士

【摘要】 鸡传染性喉气管是一种遍布世界各地的急性接触性呼吸道传染病，由传染性喉气管炎病毒（ILTV）引起，以呼吸困难、咳嗽、常咳出带血渗出物为发病特征。ILTV属于疱疹病毒科α疱疹病毒亚科，目前多用鸡传染性喉气管炎的弱毒疫苗进行预防，其中大部分弱毒疫苗株具有温和的致病性，因此可能有毒力返强的危险。 近年，ILTV基因组的大部分序列及其功能已经确定，这为基因工程疫苗的研制奠定了基础。研究表明ILTV属于疱疹病毒D型基因组，gB基因编码205KD糖蛋白复合物，能诱导体液免疫和细胞免疫，是该病毒的主要保护性抗原基因，也是研究基因工程疫苗的首选目的基因，表达gB糖蛋白是预防传染性喉气管炎的新方法。 本论文以疫苗株ILTV基因组为模板扩增出gB基因片段连接到载体DGEM-T Vector，对目的片段gB基因进行了序列测定和同源性分析，与英国Thome V882毒株、美国ILTV632毒株、澳大利亚SA 2毒株gB基因的核苷酸同源性分别为100％、100％和99.4％，氨基酸同源性分别为99.8％，99.9％和98.7％，说明它是十分保守的。然后将gB基因定向克隆于原核表达载体pBV221中构建了重组质粒pBV-gB，将其转化到受体菌DH5α中，菌株DH5α（pBV-gB）经温控诱导后，SDS-PAGE电泳可见表达的特异蛋白条带。ELISA检测说明gB基因在大肠杆菌中的表达产物具有很好的反应原性，但是鸡胚中和试验结果表明该重组菌株仅能部分中和1个EID₅₀剂量的北京株ILTV强毒。 原核表达的目的蛋白缺少生物活性和免疫原性，活载体疫苗可以克服这一缺点，因此本论文又构建了大肠杆菌一分枝杆菌穿梭表达质粒pRR-α-gB，并将其电转化至耻垢分枝杆菌，ELISA检测表明重组菌株M．smegmatis mc² 155（pRR-α-gB）的表达产物具有很好的反应原性。Western blot检测说明ILTV的gB基因在分枝杆菌中获得了表达并具有良好的免疫原性。鸡胚中和试验结果表明该重组菌株可以中和1个EID₅₀剂量的ILTV强毒，能够保护SPF鸡胚抵抗强毒攻击。它在安全性方面比弱毒疫苗更加优越，不形成潜伏感染，不会散毒。此外，该重组菌株免疫的鸡可以通过血清学方法与自然感染ILTV的鸡加以区别。因此本实验研究的工程菌M．smegmatis mc² 155（pRR-α-gB）将有希望取代现用的ILTV弱毒疫苗，彻底改变ILT防制现状。更多还原

【Abstract】 Avian infectious laryngotracheitis(ILT) is a world-wide-occurring and severe respiratory disease of chickens, which characterized by signs of respiratory depression, gasping, expectoration of bloody mucus, and high mortality. The causative agent was designated infectious laryngotracheitis virus (ILTV), or gallid herpesvirus 1, and classified as a member of the Alphaherpesvirinae subfamily of the Herpesviridae. For prevention, chickens are immunized with conventionally attenuated live vaccines. Most of these genetically uncharacterized virus strains are still moderately pathogenic, and they bear the risk of spontaneous reversion to a more virulent phenotype.As a prerequisite for the development of genetically engineered vaccines, most of the ILTV genome has been characterized by DNA sequencing over the last few years. These studies confirmed that ILTV possesses a herpesvirus type D genome. Research showed that the glycoprotein gB of is a major protective antigen. It can make body generating the antibody to eliminate infection. So gaining gB gene and its expressing product is key to the preparation of a new type vaccine. The expression of gB gene provided the basis on the preparation of vaccine and a new method to prevent chicks from ILTV infection.According to the published nucleotide suquences of gB gene of ILTV, a pair of primers used to amplify gB gene were designed and synthesized . The complete gB gene of a domestic isolation stain were amplified by PCR, and obtained about 2.6 kb fragment. A recombinant plasmid was constructed by cloning PCR product into pGEM-T vector. The sequence analysis showed that the nucleotide sequence consists of 2622 bp, encoding 874 amino acids. Comparing the isolate virus with Thome V882(England), ILTV 632 (US),and SA 2(Australia), we found that their nucleotide sequences homologies were 100%, 100% and 99.4% respectively, their amino acids sequences homologies were 99.8%, 99.9% and 98.7% respectively. The sequence comparison indicated that the gB gene of ILTV strain shares high homologies with those of strain Throne V882, strain 632 and a vaccine strain SA 2.The recombinant plasmid pGEM-T-gB was digested with EcoR I/BamH I, and the resulting fragment was inserted into the EcoR I/BamH I site of the pBV221, which was the multiple cloning sites of the vector.’ The recombinant plasmids were selected and identified byrestriction enzyme digestion which indicate a prokaryotic expression plasmid pBV-gB was constructed successfully. E.coli component host DH5 a was transformed with the expression plasmid. The recombinant bacterial stain could express the gB glycoprotein after induced by temperature, and the recombinant proteins were analyzed on 12% SDS-PAGE, and detected by ELISA with anti-ILTV serum which was prepared iii SPF chick immumized with live vaccine. The recombinant bacterial stain DH5 a (pBV-gB) can partly protect SPF chickens from a lethal dose(aEID50)of ILTV.According to the nucleotide suquences of gB gene of ILTV and the restriction enzyme digestion of the shuttle vector, two pairs of primers used to amplify gB gene were designed and synthesized. Firstly, a recombinant plasmid was constructed by cloning PCR product into pY-a vector. Then the shuttle expressive vector system was constructed by cloning the hsp- a -gB gene into the downstream sequences of pRR3 vector. The recombinant plasmid was identified by restriction enzyme digestion and electrophoreted into M.smegmatis me2 155. At last the recombinant proteins were detected successfully by ELISA and western blot, which seems to be immunogenic crucially. The recombinant bacterial stain M.smegmatis me2 155 (pRR-a -gB ) can protect SPF chickens from a lethal dose (a EID50) of ILTV. As the current commercial ILTV vaccines have several disadvantages as mentioned above, the recombinant bacterine M.smegmatis me2 155 (pRR-a -gB) can be considered very safe and therefore a promising substitution of live attenuated ILTV vaccines for future eradication of avian infectious laryngotracheitis in combination with a differential dia更多还原