Isolation And Identification Of Canine Coronavirus And Cloning Of Partial S Gene

In March 2006, two canines of police dog technical schools of the Ministry of Public Security were disease, the diagnosis that was the canine coronavirus (CCV) symptoms. Virus isolation on the dejection of these two ill dogs, we use the MDCK cells to isolate and finally isolated a strain of virus. This strain virus was observed canine coronavirus features under fluorescence microscope. So, we did the study on morphology of this virus, biological identification as well as RT-PCR, and other aspects of research. Last, the S gene from the CCV virus isolated cloned into the vector corresponding Construction to construct the recombinant plasmid. The result of this study is not only providing for the data and information on the Prokaryotic expression of CCV, but also on the development of study of CCV vaccine. The test and result is as following:1. A strain of virus was isolated from the fecal samples of a dog with clinical CCV illness. First Identification through physiochemical experiment and animal regress experiment was done. By cell culture, we found that the virus has CPE. The result of physiochemical experiment shows that the nucleotide of the virus is RNA. Physic chemically result indicate the isolate virus was sensitive to chloroform and aether, not sensitive to 1% trypsin and have a good resistant to acid, also this virus have resistant to heat. It could not be adsorbed and hemagglutinaton erythrocytes of human, some animals and chicken. The result of animal regress experiment shows that the virus can induce typical clinical syndrome of CCV illness.2. RT-PCR Identification by use of 2 pairs of common primers 2 Bp and 4 Bm and specific primers CCVF2 and CCVR2 as well as BstXâ… degestion was carried out. PCR products were purified and cloned into the pGEM-T easy Vector. The result of RT-PCR show the common primers and specific primers all get the expected amplified segment 250bp and 514bp; the result of Bst Xâ… restriction enzyme cut to the PCR Products amplified with the primers of CCVF2 and CCVR2 is appeared two amplified segment 187bp and 327bp.Recombinant plasmid Amplified result with CCVF2 and CCVR2 gained the expect amplified segment too. The result of sequencing shows that the PCR product is completely identical with that of the sequence searched in gene bank and indicated that the isolated virus is CCV.Conclusion:1. Identification through morphology, physiochemical, biological experiment, and animal regress experiment shows this virus was one strain CCV. The result of RT-PCR and Bst Xâ… digestion shows this virus was one strain CCV.2. PCR products with specific primers were purified and cloned into the pGEM-T easy Vector. The result of Recombinant plasmid and sequencing shows that Recombinant plasmid construct successful.3. We compared the sequence of this CCV S gene with other CCV strains, such as TN-449.From the results, we learned that the nucleotide sequence homology was 100%.