关键词: 犬冠状病毒大熊猫株, 核蛋白基因, 克隆, 序列分析

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