应用Pichia pastoris 酵母表达了犬冠状病毒大熊猫野毒株(CCV DXMV) S蛋白主要抗原区基因片断。用特异性引物扩增出CCV DXMV株S1基因片断，并将其克隆到pGEM-T载体中得到pTS1。用Kpn I和NotI双酶切pTS1回收目的基因S1定向克隆到pPICZαA中，构建出重组质粒pPICZαAS1。将pPICZαAS1用Sac I内切酶线性化后,电转化感受态GS115酵母细胞，用PCR法筛选阳性重组子。用1%的甲醇诱导重组酵母菌，取培养物上清进行重组蛋白的检测。结果重组酵母菌培养物上清用SDS-PAGE电泳可检测到相对分子量为106kDa大小的重组蛋白, Western-blot证实该重组蛋白可以与CCV多克隆抗体发生特异性血清学反应。凝胶薄层扫描分析表明，3株重组酵母菌在1%甲醇诱导144h后，重组蛋白S1表达量约占培养物上清总蛋白量的6.6-8.6%左右。用重组蛋白S1免疫BALB/C小鼠3次后，小鼠血清CCV中和抗体可达1:8-1:16，表明重组S1蛋白具有一定的免疫原性。

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.