关键词: 犬冠状病毒, 基因疫苗表达载体, 构建, 免疫原性

Abstract: In this experiment, three recombinant expression vectors based on pVAX1 were successfully constructed. According to the sequences of spike (S) gene, membrane protein (M) gene and nucleoprotein (N) gene of CCV Insavc-1 strain, three pairs of specific primers were designed and used to amplify S, M and N genes of DXMV strain. The PCR products were cloned into pGEM-T. Then the recombinant pTS, pTM and pTN were used for sequencing. After sequenced, three immunogenicial genes were subcloned into pVAX1, respectively, which named pVAXS, pVAXM and pVAXN. The three constructed expression vectors were transfected into MDCK cell. Transcriptions of interesting genes were detected by RT-PCR. The expression of target proteins was detected by indirect ELISA. The results showed that target genes can be transcripted after 36 hours , and target proteins can be detected after 72 hours of transformation. Immunization in dogs indicated that the three expression vectors can efficiently induce the specific cellular and humoral immunoresponse, which laid a solid foundation for the further studies of new vaccines of CCV.

Key words: canine coronavirus, gene vaccine vector, construction, immunogenicity