【作者】 杨玲；

【导师】 刘秀梵； 张如宽； 焦新安； 李厚达；

【作者基本信息】 扬州大学 ， 预防兽医学， 2002， 硕士

【摘要】 犬细小病毒（Canine Parvovirus CPV）的VP2基因编码主要衣壳蛋白，VP2基因在犬细小病毒感染过程中起着极为重要的作用，其编码CPV的主要抗原决定簇，可诱导机体产生中和抗体。 根据基因库中犬细小病毒基因序列设计合成了一对引物，其两端分别含EcoRⅠ和SalⅠ酶切位点，从犬细小病毒扬州分离株提取基因组DNA，并以此为模板，进行聚合酶链式反应（polymerase chain reaction，PCR）扩增出VP2基因的1740bp的扩增产物，将PCR产物克隆进pUC18载体，并转化大肠杆菌TG₁，通过LacZ基因的插入失活，初步筛选出重组质粒。通过限制性内切酶分析，得到CPV VP2阳性重组质粒。 对该分离株的VP2基因进行序列测定，结果表明：所扩增基因片段长度为1740bp,同美国参考株CPV-d（type 2）、CPV-15（type 2a）、CPV-39（type 2b）比较，核苷酸同源性分别高达99.5％、99.7％、99.7％，氨基酸同源性分别高达99.0％、99.7％、99.7％，表明VP2基因变异相对较少。 为探讨VP2蛋白的生物学特性，本研究还设计合成了另两对引物，其两端分别含EcoRⅠ和SalⅠ酶切位点，以扬州分离株病毒DNA为模板，扩增了VP2基因的5’端和3’端大小为794bp、1041bp的两个片段。将PCR产物按预定的阅读框架插入表达性载体pGEX-6P-1中的谷胱甘肽-S-转移酶下游，获得的重组质粒在1.0mM IPTG和37℃条件下诱导，通过对重组大肠杆菌裂解物的SDS-PAGE电泳分析，鉴定出两条分子量分别为54kD和61kD表达条带。将表达产物从SDS-PAGE凝胶中切出回收，并用其免疫6-8周龄ICR小鼠，每周免疫一次，5次免疫后，采血分离血清，所得抗血清与犬细小病毒扬州分离株感染的胎猫肾细胞（FK81）进行间接免疫荧光试验（IFA），呈现特异性荧光，表明大肠杆菌表达的VP2基因产物保留了天然VP2蛋白的抗原性。 本研究对CPV扬州分离株的VP2基因序列分析，为探索CPV抗原漂变奠定了基础；而借助于大肠杆菌表达系统制备VP2的融合蛋白，为进一步研制VP2蛋白特异单抗及筛选CPV基因工程苗创造了条件。更多还原

【Abstract】 A pair of primers were designed and synthesized based on VP2 gene of CPV from Genebank. The genomic DNA samples of canine parvovirus strain isolated in Yangzhou were extracted and used as templates for polymerase chain reaction (PCR) to amplify the VP2 gene. The specific 1740 bp band was amplifed and cloned into pUC18 vector at the restriction endonuclease EcoRI and Sail sites. The recombinant palsmids were identified by restriction endonuclease analysis. The exogenous insert of recombinant plasmid was further confirmed by sequence analysis. After sequencing, VP2 gene of Yangzhou isolation was compared with that of American reference strains CPV-d (type 2) ,CPV-15 (type2a) , CPV-39(type 2b). The results showed 99.5%, 99.7%, 99,7% homology among these different strains.VP2 fragments from CPV Yangzhou strain were amplified with another two pairs of primers. PCR fragments were cloned to the downstream of GST gene according to the right Open Reading Frame in pGEX-6p-l vector. After inducing with 1.0mM IPTG and 37℃, the inserts were expressed as two fusion proteins containing the GST protein. According to SDS-PAGE analysis, two GST-VP2 fusion proteins were found as 54kD and 61kD bands, respectively. The expressed products were extracted from the gel and added as immunogen to immunize mice intraperitoneally, 5 doses with one-week intervals. The specificity of antibody was detected positively against CPV-infected FK81 cell in the indirect immunofluorescent assay (IFA). The results of the study showed that the in vitro expressed products of VP2 gene maintained the naive antigenicity of CPV.The sequence analysis of VP2 gene will contribute to the study of antigenic drift, and the fusion proteins will be useful for the generation of specific monoclonal antibodies and recombinant vaccines.更多还原