【作者】 程丽琴；

【导师】 陈声明； 周继勇；

【作者基本信息】 浙江大学 ， 农业生物环境与能源工程， 2003， 博士

【摘要】 传染性支气管炎(Infectious Bronchitis，IB)是由传染性支气管炎病毒(Infectious Bronchitis Virus，IBV)引起的急性高度接触性传染病。是当今危害养禽业最严重的疫病之一。 IB的控制主要依赖IBV弱毒疫苗的预防接种。使用活疫苗存在外源性或垂直传播性病原污染、毒力返祖等缺陷，开发安全、有效的新型疫苗是IB控制的研究重点。近年来，用转基因植物表达抗原相对传统的疫苗生产方式来说是更经济、有效和具有发展潜力的生产系统。已经有多种抗原基因在转基因植物中表达，并且植物中表达的抗原保持良好的免疫原性，并可刺激体液和粘膜免疫。本研究在克隆和分析IBV S1遗传变异规律的基础上，构建了IBV-H52 S基因的植物表达载体，并成功转化马铃薯植物，在抗性筛选的基础上进行分子生物学鉴定，最后对植物来源的疫苗免疫动物，进行免疫原性的研究。具体结果如下： 1．参照已报道的IBV-Beaudette株S1基因核苷酸序列，设计并合成一对特异性寡聚核苷酸引物，用TRIzol试剂抽提的IBV-Sc021202毒株的病毒RNA作为反转录聚合酶链反应(RT-PCR)的模板，扩增得到长度约1.9kb的S1基因，并经测序分析。结果显示：Sc021202毒株S1基因全长为1923bp，编码S1蛋白前体，共618AA，其中，1-18个为信号肽(在真核细胞中将被切除)；19-618个为S1亚单位，共600个AA。 对获得的肾型毒株IBV-Sc021202的S1基因核苷酸及推导的氨基酸序列与GenBank中31个IBV毒株的相应序列用DNAstar、OMIGA等分析软件进行同源性比较和分析，结果表明，IBV-Sc021202毒株存在高度变异和相对保守区，含有插入和缺失突变，其中281、285和465位的氨基酸是独特的变异位点，其核苷酸与已报道的肾型毒株IBV-X、J的同源性较高，分别为94.4％和86.2％，氨基酸的同源性为94.5％和87.7％；而与肾型Holte、Gray等的核苷酸与预测氨基酸的的同源性较低，核苷酸为75.1％和72.5％，氨基酸的同源性在73.8％和73.10％；与其它毒株的核苷酸与预测的氨基酸序列的同源性在72.6-80.8％和73.1-81.8％之间。 2．用RT-PCR扩增IBV-H52S基因序列并测序分析，再利用重新设计的含XbaⅠ和BamⅢ酶切位点的引物，将获得的PCR产物定向插入植物表达载体pBI121 35S启动子下游，用三亲交配法将重组克隆导入根癌农杆菌EHA105，PCR及酶切鉴定，证实获得了含pBI121-IBVS质粒的农杆菌菌株。 3．通过农杆菌介导的遗传转化方法转化马铃薯茎段，在抗性筛选培养基上筛选，共获51株抗卡那霉素的再生植株。对所获得51个转基因马铃薯株系进行PCR检测，48株能够扩增出目的基因。 4．选取PCR鉴定为阳性的6个植株，用单一限制性内切酶酶切其基因组DNA，并进行Southern印迹杂交实验，结果表明，目的基因已经整合到马铃薯染色体上，整合的拷贝数 浙江大学博士论文为卜，大多为2拷贝。 5．对5株马铃纫’山N。，比e，n1。kt纠果，多数转杜回植株，，1。夕「源某囚能丁常转录，＊’IVJ外源插入丛囚在RNA水个上得到了表达，转录水下有差异。 6．对N；；，讨；。，；；H川为m件的枪川【11捉蛋门进行SDS十MIZ电泳和伽s比r；。印）业杂灭纠果表叫，切义个M川山泳介转从卜＋，’帆w。J状测到竹殊的蛋肉条卅，们进 步的WCs比；T；「DI迹杂交却未见I刊O蛋＊杂交条卅，排测。。｝能为及达员不够高。更多还原

【Abstract】 Infectious bronchitis (IB) caused by infectious bronchitis virus (IBV) is an acute and highly contagious viral disease of chickens. The commercial modified live and killed vaccines against the virulent IBV were available. But these conventional vaccines have many defects, such as lacking cross immunity between different serotyes of IBVs, the possibility of contamination of foreign and vertical transmissible pathogens, and the virulent recovery of vaccine virus. Therefore, it is necessary to develop safe, efficient, economic IB vaccine.The expression of antigens in transgenic plants has been increasingly used as an alternative to the classical methodologies for antigen expression in the development of vaccines. Several viral and bacterial antigens have been expressed and produced in transgenic plants. Antigens produced in transgenic plants are capable of invoking protective immune responses against important pathogens as it has already been demonstrated. Based on the analysis of genetic variantion of SI gene of IBV, the plant expression vector was constructed by fusing S gene of IBV-H52 with pBI121. Potato plants were genetically transformed with cDNA construct encoding the full-length glycoprotein S of IBV under the control of the cauliflower mosaic virus 35S (CaMV 35S) promoter by the Agrobacterium system. Transgenic potatoes screened on (he media containing kanamycin and carbenicillin were analyzed by PCR and Southern, Northern and Western blot.The main results presented in this thesis are as follows:1. The specific primers were designed and synthesized according to the published sequence of SI gene of IBV-Beaudette strain. cDNA of SI gene were amplified by reverse transcription polymerase chain reaction (RT-PCR) from viral RNAs of IBV-SC021202 isolate, which was extracted with Trizol reagent. The PCR products about 1.9kb in length were cloned into pGEM-Teasy vector for sequencing. Sequencing results showed that SI gene of the nephropathogenic isolates IBV-SC021202 was 1855bp in length and encode a protein of 618 amino acids which consist a signal peptide (1-18aa) that were to be cut of in eukaryotic cell and SI subunitof600aa(19-618aa).The nucleotide and deduced amino acid sequences of the SI gene of IBV Sc021202 isolate were compared with those of the other 31 IBV strains from GenBank with such softwares as DNAstar, OMIGA.The result showed that there are HVR and relatively conserved region in it. Insertion and deletion were both found in it and the mutation at 281,285 and 465 aa were unique. The SI gene of IBV SC021202 isolate showed high homology with that of nephropathogenic IBV X and J strains (94.4% and 86.2% in nucletide sequence and 94.5% and 87.7% in amino acid sequence), low homology with that of nephropathogenic IBV Holte strain and Gray strain (75.1% and 72.5% in nucletide sequence and 73.8% and 73.1% in amino acid sequence), and relatively constant value of homology with the other IBV strains (72.6% and80.8% in nucletide sequence and 73.1% and 81.8% in amino acid sequence).2. The ORF of S gene of IBV H52 strain, encoding a peptide composed of 1162 amino acid residues, was cloned into the plant expression vector BI121 under the control of 35S promoter. PCR and restriction analyses confirmed that the recombinanl plasmids containing the target gene were obtained. Recombinant plasmids were transferred into Agrobacterium tumefaciens EHA105 by tri-parenlal mating method. PCR and restriction detection also confirmed that the target gene was transferred into Agrobaclerium tumefaciens.3. Different potato plants were genetically transformed with cDNA construct encoding the full-length glycoprotein S of IBV, responsible for the neutralizing antibody induction against the virus, under the control of the cauliflower mosaic virus 35S (CaMV 35S) promoter by the Agrobacterium tumefaciens mediated system. The plantlets grown on media containing kanamycin and carbenicillin were analyzed by PCR and found that IBV-S gene could be amplified from 48 of 51 (94%)更多还原