【作者】 莫小见；

【导师】 郭爱珍； 陆承平；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2004， 硕士

【摘要】 CpG DNA作为一种新型的免疫佐剂是目前分子免疫学研究的热点之一。CpG DNA是含有CpG基序（CpG motifs）的细菌DNA、质粒DNA和寡核苷酸（ODN）的统称。CpG DNA具有很强的非特异性免疫刺激作用，可在无T细胞存在的情况下直接激活B细胞，亦可直接激活单核细胞、巨噬细胞及树突状细胞等免疫细胞。早期研究指出，来自多种哺乳动物的细胞可被CpG DNA激活，但是对不同免疫对象而言，具有最佳免疫刺激作用的CpG基序是不同的。因此，筛选各种动物各自的最佳CpG基序对于新型疫苗佐剂的研究开发具有极大理论价值和实用意义。国外已经有关于人和小鼠的最佳基序的研究报道，国内关于CpG的基础性研究尚少，且未见有关犬的最佳CpG基序的研究报道。 犬瘟热（Canine distemper，CD）是对犬等食肉目动物危害最大的病毒性传染病之一，而犬瘟热病毒（Canine distemper virus，COY）NJ-15株则是南京地方分离株。为了探讨CDV NJ-15株的致病机理，研制免疫保护率高、保护期长的犬瘟热基因工程疫苗，本研究对与CDV NJ-15株致病性和免疫原性密切相关的血凝素蛋白（H）基因进行了克隆、序列分析，分别构建了原核表达载体和真核表达载体，并对表达产物的免疫原性进行了初步研究。另外，本研究进行了刺激犬B淋巴细胞分泌IgM的最佳CpG基序的体外筛选试验，为CDV基因工程疫苗的研制提供有的放矢的新型佐剂材料。 1 犬瘟热病毒NJ-15株H基因的T-A克隆与序列分析 将CDV NJ-15株感染Vero细胞收获病毒，参照RNAgents^R Total RNA Isolation System（Promega公司）的说明，提取CDV NJ-15株基因组RNA为模板，采用反转录聚合酶链反应（RT-PCR），扩增到包含H基因全长约1.9Kb的基因片段。将RT-PCR产物T-A克隆到pMD 18-T载体中，经蓝白斑和酶切分析筛选到阳性克隆载体命名为pMD-H。并对CDV NJ-15株H基因的序列进行分析。 2 犬瘟热病毒NJ-15株H基因片段的克隆和表达 对克隆载体pMD-H进行双酶切，将得到的1058bp片段以正确的阅读框架定向克隆于pET-28b（+）中。将重组质粒转化进宿主菌Rosctta^TM中，在37℃ 1mM IPTG诱导下该片段获得良好的表达。经SDS-PAGE鉴定其表达的融合蛋白质约38KDa，与预期值一致。免疫转印试验显示，体外表达的该重组蛋白可被CDV参考株Onderstepoort兔抗血清识别，表明该重组蛋白具备天然H蛋白的部分抗原表位。pET28-H虽然未表达了H蛋白的全基因，然而免疫转印证实其仍然具有某些天然蛋白的关键性表位，为研制CDV的亚单位疫苗提供了候选材料。犬应热病.南京株（N十1 5）H签因的克隆、裹达以及弱傲犬B淋巴细胞分泌l脚的.佳饰口共序的体外娜选3犬应热病毒NJ一15H基因片段的克隆和及其在真核细胞中的瞬时衰达 对克隆载体pM}H进行双醉切，将得到的cDV血凝素蛋白（H）基因插入含有以V启动子的pcDNA3.1/Zeo质粒载体中，通过脂质体介导转染COS一细胞，用SDS一PAGE和免疚转印分别鉴定表达产物的相对分子量及其免疚特性.结果证明获得正向插入的PcONA一H重组表达质粒，表达产物的相对分子1约77kDa，与预期值大小一致，且该表达产物能够被CDv参考株Dnder:tepo。rt的兔血清识别，表明构建的pcnN^一H重组质粒能在哺乳动物细胞中表达并且具有抗原性，该重组质粒有可能用作CDv的DN人疫苗.4刺激犬B淋巴细胞分泌l脚的最佳饰G ODN的体外筛选 制备犬B淋巴细胞，进行体外培养，以人工合成的24种CpG ODN分别刺激之，48h后收集培养上清，ELISA检测IgM.发现第12号cpG ODN（5’ggGGcT旦工旦旦工工TcTG“993’）具有最佳的免疫刺激性，与国外学者断近报道的结果相符，为cDv的。NA疫苗提供了可供选择的位剂.更多还原

【Abstract】 Recent studies showed that CpG DNA, such as bacterial DNA, plasmid DNA and oligodeoxynucleotides (ODN) which all contain unmethylated CpG motifs, could be used for novel vaccine adjuvant to enhance the humoral and cellular immune responses. It was reported that CpG DNA can activate plasmacytoid dendritic cells (pDCs) and B cells directly, which express TLR9, and secondarily activate monocytes^nacrophages, NK cells, T cells and some non-immune cells, improve the ability of neutrophils to provide effective host defense, and produce Thl-like proinflammatory cytokines, interferons, and chemokines, to activate host defense mechanisms leading to innate and acquired immune responses. Therefore ,CpG DNA is expected to be a novel adjuvant for prophylactic and therapeutic vaccination for cancer, allergy and infectious diseases. However, one certain CpG motif is not always optimal to all host, so it is very important to screen CpG ODN with strong immunostimulatory activity on canine in vitro.Canine distemper (CD), a highly contagious disease with worldwide distribution, could cause severe economic losses to the canine fanning, far cultivation and wildlife conservation. The causative agent of the disease, canine distemper virus (CDV), is a member of Paramyxoviridae whose genome is a non-segmented single-strand negtive-sense RNA. The CDV NJ-15 strain is a virulent CDV isolated from dogs in Nanjing. Traditional attenuated vaccine against CDV has intrinsic shortcomings despite its important role in controlling the occurrence of CD. It can cause immunosuppression and lesion of CNS, and it may be lethal some susceptible animal and possess a wild hot spectrum. Moreover, mutation of CDV may also cause epidemic of CD. In order to develop a new kind of safe, efficient and convenient CDV genetically engineered vaccine, we constracted one genetic recombinant eukaryotic expression plasmid pcDNA-H and another recombinant prokaryotic expression plasmid pET-H, which express the H gene or its fragments, with the Nanjing isolate of CDV on the initiative exploring of the molecular property of H protein that is tightly related to the pathogenicity and immunity of CDV andsubsequently detected their protection immunogenicity in mice and dogs. These above works can be divided into 4 parts:1 T-A Cloning and Sequence analyses of hemagglutinin protein gene of Nanjing isolate Virus was isolated from the CDV NJ-15 strain infected Vero cells to extract its RNA as template of RT-PCR, and then we amplified the full-length 1815bp hemagglutinin protein (H) gene . The PCR products were inserted into pMD 18-T vector routinely, and the positive recombinants were identified by blue-white screening and endonuclease digestion. H gene we got were then sequenced and analyzed, the nucleotide and predicted amino acid of Nanjing isolate is 99% identical to the vaccine strain-Onderstepoort stain of CDV. Comparison from other isolates shows the 90~96% identity respectively. Amino acid alignment shows that there are only 4 potential asparagines glycosylation site in the protein,while there are 8-9 in other isolates, such as Changchun isolate, American isolat and Japan isolate.etc.2 Cloning and Expression of the Fragment of Canine Distemper Virus Haemagglutinin(H) Protein GeneThe 1058bp fragment of H gene of CDV NJ-15 was gained by digesting the plasmid pMD-H with EcoR I and 5a/ I, then it was cloned into the expressing plasmid vector pET-28b(+).The recombinant was transformed into the host strain Rosetta?and induced to express by l.OmM BPTG at 37C.The expression product was identified by SDS-PAGE and found to be 38KDa as expected one and confirmed by Western blot with CDV Onderstepoort strain antiserum. The results revealed that in vitro expression of the fragment of H gene had the critical antigenitic epitopes of CDV.3 Cloning and Transient Expression of the Full-length Gene Encoding the Nanjing strain of Canine Distemper Virus Haemagglutinin(H) protein in Mammalian CellsThe H protein gene was gained by digesting the plasmid pMD-H w更多还原