Amplifying,Cloning And Sequencing Of S Gene Of Canine Coronavirus Isolated From Giant Pandas

Three giant pandas were ill in the zoo of Chongqing in 1997, and two died after some treatments. Virus isolation and identification were adapted on livers and spleens of the two died pandas. Finally he isolated a strain of virus named DXMV. Cell cultivatings of virus were examined by negative staining electron microscope, and coronavirus-like particles were found. Then partial spike protein gene of the virus was amplified by RT-PCR with the primers which could amplify 11 kinds of coronaviruses. The result indicated that it belonged to a kind of coronavirus. They designed a pair of primers in S gene region according to CCV K378 strain's sequence, and amplified a target fragment of S gene from 81st to 1166th. This strain was identified to be canine coronavirus by gene sequencing and comparing. The study aimed at amplifying, cloning and sequencing S gene of DXMV, and analyzing homology with other CCV strains.CCV only has one serum type, but virulence of various strains from different regions and animals are different. Cloning and sequencing of virus gene are the best methods of analyzing genetic character of virus. S protein which is encoded by S gene is the greatest protein in the virus, composing of 1452 amino acids, is a relatively conserved protein. It can combine with cell receptor and stimulate body to produce neutral antibody. Therefore, S gene is a major determined factor that has pathogenic and host specialty. However, studies are less about S gene and gene variation. The major content of the research is to sequence S gene of CCV, to study its molecule biology character, and to discuss its difference with other strains. The research will offer scientific materials for studying CCV's gene-engineering vaccines, and also make a basement for giant panda's prevention and cure of virus disease.It is difficult to amplify the whole S gene with a pair of primers, because the virus can not multiply in the cell greatly. So we designed nested primers to amplify Sgene for five fragments according to the sequence of CCV K378 strain published in GeneBank. Six primers were designed with the help of Primer Premier 5.0 software. They were SF2-SR2, SF3-SR3, SRI3 and SFI3. SF2-SR2 amplified a fragment of 792bp; SF3-SR3 were outer half-nested primers, and SRI3 and SFI3 are inner half-nested primers. They amplified two gene fragments of 737bp and 1178bp separately. Meanwhile, primers SFi-SRi, SF4-SR4 and SFLt were designed in terms of CCV Insavc-1 strain. SFi-SRi amplified a fragment of 368bp; SF4-SR4 and SFI4 could nestedly amplify a gene fragment of 985bp.The PCR products were purified and then cloned into pGEM-T vectors. Recombinant plasmids were identified by ways of PCR and restriction endonuclease digesting, and finally were sequenced. The five sequences were edited to be a whole sequence of S gene. Using bio-software, S gene sequence of DXMV was compared with those of other CCV strains, such as CCV K378, CCV V54, CCV 23/03, etc. The results showed that homology of the nucleotide sequence was 99.5%, 94.2% and 56.9% respectively, and homology of the deduced amino acid sequence was 99.0%, 96,1% and 50.0%.In this study, the S gene of CCV which was isolated from giant pandas was cloned, sequenced and analyzed. The result of the study not only provided some necessary information for panda's disease controlling work, but also enriched the research contents of coronavirus .