犬冠状病毒核酸探针的制备及其基因序列的测定与比较

Development of Nucleotide Probe to CCV and Comparison of the Sequences

采用 RT- PCR方法扩增犬冠状病毒 (CCV) YS1、CI1和 NL- 1 8株 5′端部分 S基因序列 ,以随机插入 DNA法对CCV YS1株纯化的 PCR产物标记 32 P同位素 ,制备核酸探针 ,并与 3株 CCV反转录产物杂交。用平端连接法将 3株CCV PCR产物克隆于 p U C1 9Sma 位点或 p GEM- T载体中 ,经 PCR鉴定为正确重组质粒。以双脱氧末端终止法测定了重组质粒的 c DNA核苷酸序列 ,并用 DNASIS计算机软件进行多重比较分析 ,绘制系统树。结果表明 ,所制备的核酸探针可与 3株 CCV反转录产物杂交 ,其核苷酸序列与多株 CCV相应序列的同源性高达 91 .9%～ 99.1 % ,为CCV的保守区。由此说明 ,制备的核酸探针可用于犬

Partial spike protein genes of canine coronavirus(CCV) YS1,CI1 and NL 18 strain were amplified with the method of RT PCR and the purified PCR product of CCV YS1 strain was labelled with 32 P isotope to develop the nucleotide probe to CCV.And the RT products of the 3 CCV strains were detected with the probe.Then the 3 PCR products were cloned into the Sma I point of pUC19 or pGEM T vector,sequenced by the Track Sequencing System and compared with the DNASIS Computer Software.The results showed that the probe could detect the RT products of the 3 CCV strains and the sequences have 91 9% 99 1% homology with that of the other CCV stains.The nucleotide probe sequence is conserved according to the result of comparsion,so it could be used to study the molecular epidemiology of CCV infection of dogs.