犬冠状病毒V1株核蛋白基因的克隆与序列分析

首次对犬冠状病毒V1野毒株核蛋白(N)基因进行了克隆和序列测定。根据GenBank中报道的CCV Insavc-1株N基因序列，设计了1对特异性引物，对分离的CCV V1野毒株进行了RT—PCR扩增；将扩增得到的阳性PCR片段经纯化后与pGEM—T连接得到重组质粒pTN1，进行核苷酸序列测定，并与GenBank中CCV标准毒株Insavrc—1 N基因进行了比较。结果该基因全长为1146bp，编码382个氨基酸，两者核苷酸的同源性为91．7％；推导的氨基酸序列的同源性为90．2％，显示出较高的保守性。在V1野毒株推导的N蛋白N端156-179位存在一个SRXX富集区，与小鼠肝炎病毒相应区域相同，推测可能是RNA结合区。另外，推导的该蛋白氨基酸序列其疏水性和抗原表位与标准毒株Insavc-1 N蛋白存在一定的差异；在氨基酸组成上，该蛋白丝氨酸和赖氨酸含量高达9．84％，提示该蛋白具有高度螺旋和缠绕的分子结构：

CLONING OF NUCLEOCAPSID PROTEIN GENE OF CANINE CORONAVIRUS STRAIN V1 AND ITS SEQUENCE ANALYSIS

Nucleocapsid protein gene of canine coronavirus, strain VI, has been cloned and sequenced in China. According to the nucleocapsid protein gene sequence of canine coronavirus strain VI reported by Genbank, a pair of specific primers was designed and used to amplify N gene. The positive PCR product was purified and ligatured with pGEM-T. The correct positive recombinant was used for sequencing. The length of N gene of CCV V1 was 1 146 bp and it encoded 382 amino acids. The homology of nucleic acids and amino acids between CCV V1 and Insavc-1 was 91.7% and 90.2%, respectively, suggesting that they are considerably conservative. A region, deduced to be the RNA binding region rich in SRXX, was found, which shared the same site with MHV, deduced to be the RNA binding region. Some differences were observed between the two strains in their hydrophobicity and antigenic index. The N protein had high contents of Ser and Lyx (9.84%), suggesting that it molecular structure was a highly spiral helix.