【作者】 乌仁高娃；

【导师】 陈振文；

【作者基本信息】 中国人民解放军军事医学科学院 ， 预防兽医学， 2004， 硕士

【摘要】 犬瘟热病是由犬瘟热病毒(CDV)感染犬和其他食肉动物的多发性、致死性传染病。CDV属于副粘病毒科麻疹病毒属，为有囊膜的负链线性RNA病毒，基因组长约16kb。基因组中包括6个非重叠基因区。它们的编码基因按3′～5′端顺序依次为N-P-M-F-H-L系列。最新研究表明CDV融合蛋白(F)和血凝素(H)蛋白是宿主免疫系统的主要目的抗原，是产生中和抗体的重要抗原之一。核衣壳(N)蛋白是保守性较强的免疫原性蛋白，此外N蛋白上含有T细胞表位。H，F和N蛋白在犬瘟热的免疫预防方面起重要作用。 本研究旨在获得CDV的N，F和H蛋白基因，将其克隆后在大肠杆菌中表达，获得大量易于纯化的N，F和H蛋白并对其功能进行研究，为研制基因工程疫苗和诊断试剂等打下理论基础。 一、CDV N基因保守区序列(NC)的克隆及表达：从犬瘟热病毒Onderstepoort株中提取病毒RNA，经反转录聚合酶链反应(RT-PCR)获得犬瘟热病毒全长N基因，然后应用2对引物扩增获得2段CDV编码N蛋白保守区基因(NC1和NC2)，将此片段克隆到质粒pGEM-Teasy测序，测序结果显示NC1和NC2与CDV Onderstepoort株DNA和推导的氨基酸同源性分别为99.7％、99.3％和100％、100％，利用谷胱苷肽巯基转移酶(GST)融合表达载体pGEX-5x-3和麦芽糖结合蛋白(MBP)融合表达载体pMAl-C2构建了重组表达载体pGEX-5x-3-NC2和pMAl-C2-NC1，重组表达质粒经PCR和酶切鉴定后，将其分别转化到大肠杆菌BL21(DE3)plys中和DH5α细胞中，经IPTG诱导，pGEX-5x-3-NC2没有获得目的蛋白表达，而pMAl-C2-Nc1成功获得了目的蛋白表达。 二、CDV H基因的克隆及表达尝试：应用一对引物，经RT-PCR扩增获得CDV全长H基因，将此片段克隆到pGEM-Teasy测序，测序结果显示与CDV Onderstepoort株DNA同源性为98.8％，所编码的氨基酸同源性为98.2％。然后利用6×His标记的重组表达载体pQE-32构建了重组原核表达载体pQE-32-H，重组表达质粒经PCR和酶切鉴定后，转化到大肠杆菌M15中，经IPTG诱导，没有获得目的蛋白的表达。 三、CDV F基因的克隆和表达：应用一对引物，经RT-PCR扩增获得CDV全长F基因，以全长F基因为模板应用4对引物，通过重叠PCR(OE-PCR)扩增获得缺失疏水区的F基因(dF)。将此片段克隆到质粒pGEM-Teasy测序，测序结果显示，与CDVOnderstepoort株DNA同源性为99.2％，所编码的氨基酸同源性为98.4％。将此片段插入 中文摘要到pQE一30质粒构建了重组pQE一30一dF原核表达质粒，重组表达质粒经PCR和酶切鉴定后，转化到大肠杆菌M巧细胞中，经IPTG诱导，获得了目的基因的高效表达，经聚丙烯酞胺凝胶电泳和W己stem一印迹试验，验证其表达的重组蛋白产物分子质量大小为预期的47000。将表达产物用Ni一NTA琼脂糖进行纯化，获得较高纯度的目的蛋白，目的蛋白进行复性后免疫小鼠，所得的抗血清与CDV感染的Vero细胞在免疫荧光试验(IFA)中，呈细胞阳性染色。 四、CDVF蛋白的应用:以重组表达的F蛋白作为包被抗原，初步建立了检测犬瘟热血清抗体的间接ELIsA方法。所建立的间接ELISA抗原包被浓度为125n留孔，血清最佳稀释度为1:160。经阻断试验、重复性试验等表明该方法特异性强、重复性较好，可用于犬瘟热血清抗体的定量和定性检测。更多还原

【Abstract】 Canine distemper caused by canine distemper virus (CDV) is a highly infectious, frequently lethal disease with high mortality in dogs and other carnivores. CDV is belong to RNA genus morbillivirus in the paramyxoviridae family that has envelop, a single-stranded, negative sense gnome of approximately 16kb. The genome contains six non-overlapping gene regions, organized 3 N-P-M-F-H-L-5.Many studies have shown that two glycoproteins (Hemagglutinin H and Fusion F protein) of CDV are major target antigens for the host immune system. Nucleocapsid (N) protein is highly conserved immunogenicity protein. In addition, N protein contains T cell epitope. Hemagglutinin protein, Fusion protein and Nucleocapsid protein play an important role in immuno-prevention of canine distemper.This reseach is amied at cloning and expressing N?F and H gene of CDV and obtaining the easily purified expression products of N?F and H gene of CDV, it would help to study of gene vaccine and diagnostic reagent.1.Cloning and expression of CDV NcCDV RNA was extracted from CDV-Onderstepoort strain. The full length N gene was amplified by RT-PCR and the conserved gene of N (Nc) was amplified by PCR with two specific primers. This sequence was cloned into plasmid pGEM-Teasy. Both the DNA sequence of the cloned gene and amino acid sequence have identity of 99.7%, 99.3% and 100%, 100% with CDV Onderstepoort strain. The two Nc genes were cloned into GST fusion-expression plasmid pGEX-5x-3 and MBP fusion-expression plasmid pMAL-C2, respectively, to construct recombinant plasmid pGEX-5x-3-Nc2 and pMAL-C2- Ncl. The recombinant plasmid were verified by PCR and restriction endonuclease analysis. The recombinant plasmid pGEX-5x-3-NC2 and pMAL-C2- Ncl were transformed into the BL21(DE3)plys and DH5?respectively, and induced by IPTG. As a result, recombinant protein of pMAL-C2- Ncl was obtained, however, the pGEX-5x-3-NC2 recombinant protein could not expressed.2.Cloning and expession of CDV H geneThe full length H gene of CDV was amplified from CDV RNA by using RT-PCR with apair of primers, then this fragment was cloned into pGEM-Teasy. The DNA sequence of the cloned gene and the deduced amino acid sequence showed an identity of 98.8% and 98.2% with Onderstepoort strain, respectively. The recombinant expression plasmid pQE-32-H was constructed by using pQE-32 signed with 6 X His. The recombinant expression plasmid was verified with PCR and restriction endonuclease analysis before transformed into E.coli Ml5. The plasmid was induced with IPTG, however, the intrerest protein was not obtained.3.Cloning and expressin of CDV F geneThe full length F gene of CDV was amplified from CDV RNA by using RT-PCR with a pair of specific primers, then using the full length F gene as template, the deleted hydrophobic domains of F gene (dF) was amplified by overlap extension -PCR(OE-PCR)with four specific primers and this fragment was cloned into pGEM-Teasy and sequenced.Sequence analysis showed that the DNA sequence of cloned gene and the deduced amino acid sequence has identity of 99.1% and 98.4%,respectively.The dF fragment was cloned into pQE-30 to construct a recombinant expressing plasmid pQE-30- dF. The recombinant plasmid was verified by PCR and restriction endonuclease analysis, then it was transformed into E.coli strain Ml5 and induced by IPTG and obtained high level expression of aimed protein. The expression product dF gene was identified by SDS-PAGE and western-blotting, and found to be 47000 in size as a fusion protein. The protein was purified by Ni-NTA agarose and recovered. The purified product of dF protein was injected into the mice every two weeks and for four times. The antiserum was collected from the immunized mice and used for immunity fluorescence assay (IFA) with Vero monolayer infected by CDV. The positive staining was found and localized on the infected cells.4. Application of CDV F proteinAn indirect ELISA method for measuring antibody of canine distemper virus (CDV) was established with recombinant fusion protein (F) of CDV as antige更多还原