| Canine distemper (CD), caused by canine distemper virus (CDV), is acute and highly contagious disease in canine and other carnivores, and is one of the most severe infectious diseases in canine farming, far cultivation and wildlife conservation. Traditional attenuated vaccine has intrinsic shortcomings despite its important role in controlling the occurrence of CD. In order to develop a new kind of safe and efficient CDV genetically engineered vaccine, we cloned and sequenced the Fusion (F) protein gene of CDV witch was isolated by ourselves and named CDV-SJ, and constructed recombinant eukaryotic expression plasmid pcDNA3, 1-F, which was detected if express the F gene fragment. The examination will set up foundation for construct new kind of safe and efficient CDV genetically engineered vaccine.1. One pair of primers was designed and synthesized based on the Fusion (F) protein gene sequence of the Onderstepoort strain of CDV. The templates were gained by the reverse transcription (RT) reaction from total RNA that was extracted from the Vero cells infected with the isolated CDV. The F gene fragment was amplified by polymerase chain reaction (PCR). A 1053 bp fragment of the PCR production was connected with the vector pMD18-T, and then the positive recombinant plasmid was identified by ampicillin screening and endonuclease digestion. The correct positive recombinants were sequenced. The open reading frame length of the F gene of CDVSJ was 1053bp and encoded 351 amino acids. The CDVSJ in comparison with other CDV isolated strain--00-2601 USA strain, 01-2689 USA strain, MS01 China strain, ONP strain, A75-17 strain, PDV-2 strain, DOGDK 91C strain shows that the homology of nucleotide sequence is 95.4%, 94.4%, 93.3%, 97.3%, 94.5%, 94.7%, 93.9%, respectively. The CDVSJ in comparison with 00-2601 USA strain, 01-2689 USA strain, MS01 China strain, ONP strain, A75-17 strain, PDV-2 strain, DOGDK 91C strain shows that the homology of predicted amino acid is 95.4%, 94.4%, 93.3%, 97.3%, 94.5%, 94.7%, 93.9%, respectively. But the antigen point between CDVSJ and ONDER strain, 00-2601 USA strain, MS01 China strain, PDV-2 strain have the biggish difference in position of 40-60 amino acids. The antigen point of the ONDER strain and 00-2601 strain were same in the 110-120 amino acids and another antigen point were same but witch of them were lower than the formers. Only the antigen point of PDV-2 strain had different in the 120-135 amino acids to the others. The antigen point of CDVSJ had different in the 135-140, 190-200 and 340-351 amino acids to the others. These results indicated the immunity defeat may be caused from forecast latent antigenic determinant differences between the isolated strain and vaccine ONP.2. The F gene was digested from the recombinant plasmid pMD 18-T-F with BamH I and Kpn I, and then the fragment was cloned into the eukaryotic expression vector pcDNA3.1 (+). The positive recombinant plasmid pcDNA3.1-F was transfected into mammalian BHK-21 cell by fluorescent antibody and RT-PCR were used to detect transient expression of the recombinant eukaryotic expression plasmid pcDNA3.1-F in the BHK-21 cell. A 1053 bp fragment of the specificity F gene was obtained by RT-PCR appraisal response after total RNA abstraction. The results showed the positive recombinant plasmid harbour the right inserted F protein gene was obtained. The positive recombinant plasmid pcDNA3.1-F transfecting the BHK-21 cell presents specificity yellowish green fluorescence by indirect fluorescent antibody detects. But the BHK-21 cell was transfected by the idling pcDNA3.1 (+) and the normal cell has non-specificity yellowish green fluorescence. All of them indicate pcDNA3.1-F...
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