Construction of Canine parvovirus DNA Vaccine and Detection of Its Inoculated Mice

用PCR方法扩增犬细小病毒(Canine parvovirus，CPV) 貉分离株(PV/貉/CC/1/86)的VP2蛋白基因，并将其克隆入pMD18-T，命名为pTCPV，进行测序分析。结果除发现300G→S突变外，还发现32G→D突变。然后将PV/貉/CC/1/86 VP2蛋白基因克隆入真核表达载体pVAX1的CMV启动子的下游，构建了CPV核酸疫苗的真核表达质粒pVCPV。pVCPV转染BHK-21细胞能够表达CPV VP2蛋白，所表达的CPV VP2蛋白能够与抗CPV的阳性抗体发生特异性的抗原-抗体反应。用pVCPV免疫接种小鼠，用ELISA和微量中和试验检测免疫小鼠抗CPV抗体阳性，但是没有检测到抗CPV HI抗体； pVCPV免疫小鼠的脾淋巴细胞对特异性和非特异性抗原刺激均有明显的增殖。结果表明, pVCPV免疫接种小鼠能够诱导机体产生抗CPV的特异性体液免疫和细胞免疫。

Construction of Canine parvovirus DNA Vaccine and Detection of Its Inoculated Mice

The VP2 gene of Canine parvovirus (CPV) isolated from a raccoon dog was amplified by PCR and cloned into the vector pMD18-T. The recombinant, named pTCPV, was sequenced and analyzed. As a result, a G→S substitution at amino acid residue 32 was observed besides of 300 G→S. Then, the VP2 gene was sub-cloned into the vector pVAX1 at the downstream of CMV. The recombinant, named pVCPV, was used as CPV DNA vaccine. BHK-21 cells transfected with pVCPV did express CPV VP2 protein specifically reacting to anti-CPV sera. The mice vaccinated with pVCPV developed both anti-, CPV ELISA antibody and SN antibody, but was not detected anti-CPV HI antibody. Lympho-spleencytes of the inoculated mice was proliferated with CPV and ConA stimulation, respectively. The results indicate that the mice inoculated with pVCPV develops the humoral immunity and cellular immunity against CPV.