Abstract: Nucleoprotein (N) gene of canine coronavirus giant panda isolate (CCV GP) was firstly cloned and sequenced. According to nucleoprotein gene sequence of CCV strain Insavc-1 accessed by GenBank, a pair of specific primers was designed to amplify N gene of CCV strain GP. The PCR product was purified and cloned into pGEM-T. The positive recombinant pTN was used for sequencing. The complete length of N gene of CCV GP was 1 146 bp, which encoded 382 amino acids. The homologies of nucleic acid and amino acid between CCV strain GP and Insavc-1 were 92.6 % and 93.2 %, respectively. A region abounding with SRXX was found, which located in the same region with that of MHV. It was deduced to bind genomic RNA. There were some differences in the hydrophobicity and antigenic index plots between two strains. The N gene was further subcloned into the prokaryotic expressing vector pET28a and then transformed into E.coli strain BL21 for expression under the induction of IPTG. The recombinant protein was identified by SDS - PAGE and Western-blotting analysis. The results revealed that it had a molecular weight of 48 KD, which could be specifically recognized by multiclonal antibody against CCV. The recombinant protein N can account for 49.3 % in the total protein of the induced recombinant bacteria by analysis of gel scanning, which can be used for antigen to detect specific antibody against CCV in giant panda's sera.

Key words: Canine coronavirus giant panda isolate, Nucleoprotein gene, Cloning, Sequence analysis

摘要： 首次对犬冠状病毒大熊猫株(CCV GP) 核蛋白(N) 基因进行了克隆和序列测定。本实验根据GenBank 中报道的CCV lnsavc - 1 株N 蛋白基因序列, 设计了一对特异性引物, 对分离的CCV GP 野毒株进行了RT - PCR 扩增。将扩增的PCR 产物纯化回收后与pGEM-T 连接得到重组质粒pTN , 进行核苷酸序列测定。结果该基因全长1 146 bp , 编码382 个氨基酸; 与CCⅤ标准毒株Insavc -1 N 基因相比, 核苷酸的同源性为92.6 % , 推导的氨基酸的同源性为93.2 %。在推导的N 蛋白N 端156 - 179 位存在一个SRXX富集区, 与小鼠肝炎病毒N 蛋白相应区域相同, 推测可能是RNA 结合区。预测的GP 株N 蛋白疏水性和抗原表位与Insavc - l 株N 蛋白存在细微的差异。将pTN 双酶切, 回收目的基因片段克隆到大肠杆菌表达载体pET28 a 中构建了重组质粒pETN , 转化大肠杆茵BL21 ,用IPTG进行了诱导表达。结果重组菌菌体裂解物经SDS - PAGE 电泳可检测到相对分子量为48 KD 的重组蛋白,免疫印迹法证实该重组蛋白可以与CCV 多克隆抗体发生特异性反应。经凝胶薄层扫描分析, 重组N 蛋白表达量可占菌体蛋白的49.3 % , 表达的蛋白纯化后可用于建立检测大熊猫CCV 抗体间接ELISA 用的包被抗原。

关键词: 犬冠状病毒大熊猫株, 核蛋白基因, 克隆, 序列分析