Expression of the Major Antigenic Region Fragment of Spike Gene of Canine Coronavirus and Immunogenicity of Expressed Products

In this study, the major antigenic region of spike (S) gene of Canine coronavirus giant panda’s isolate (CCV strain DXMV) was expressed in Pichia pastoris. The S1 gene fragment encoding major antigenic region of S protein was amplified and cloned into T vector, then subcloned into pPICZαA for yeast expression. The recombinant pPICZαAS1 was linearized with SacI and then transformed into competence GS115 cells for expression under the induction of 1% methanol. The positive yeasts were screened by PCR. Expression of S1 protein was identified by SDS-PAGE and Western blot. The results revealed that it had a molecular weight of 106 kDa, which could be specifically recognized by multiclonal antibody against CCV. The expression of recombinant S1 protein amounted to 6.6-8.6% of the total protein of supernatant by gel scanning. After three immunization by the recombinant S1 protein, the neutralizing antibody in sera of mice can range from 1:8 to 1:16, which indicated that the recom- binant S1 protein was of it’s immunogenicity.