以CpG为佐剂的犬细小病毒基因疫苗的初步研究

目的研制犬细小病毒(CPV)基因疫苗。方法以CPV VP2基因为基因免疫的目的基因,以pcDNA3和pcDNAK质粒为基因免疫的载体,以非甲基化的胞嘧啶鸟嘌呤二核苷酸(CpG)为核心的免疫刺激序列为免疫佐剂,构建重组质粒并免疫BALB/c小鼠和毕格犬。结果经pcDNA3-VP2C1(含1个拷贝CpG基序)基因免疫的BALB/c小鼠能产生抗CPV血凝抑制抗体;对于经CPV灭活苗初次免疫的毕格犬,用pcDNAK-VP2C2(含2个拷贝CpG基序)质粒免疫产生的再次免疫应答优于pcDNA3-VP2C1。结论VP2基因、pcDNAK和犬源CpG可用于CPV基因疫苗的进一步研究。

Development of a Gene Vaccine Containing CpG against Canine Parvovirus Disease

Objective To develop a DNA vaccine against canine parvovirus(CPV) infection.Methods The VP2 gene of CPV with or without CpG motif was cloned by PCR.The eukaryotic expression vector pcDNA3 was modified by restriction digestion to remove its neo~r gene and to replace its amp~r with kan~r to conform gene therapy requirements.The recombinant plasmids were constructed by subcloning the VP2 gene with or without CpG motif into the modified vector pcDNAK and injected into BALB/c mice and Beagle dogs.Both primary and secondary antibody responses were detected by hemagglutination inhibition(HI) assay.Results Injection of the recombinant vector pcDNA3-VP2C1 containing one copy of the consensus CpG motif into BALB/c mice lead to more elevated antibody response than the empty vector pcDNAK and the recombinant vector pcDNAK-VP2 without CpG motif.Gene immunization experiments in dogs preimmunized with an inactivated vaccine showed that the pcDNAK-VP2C2 vector containing two copies of canine-specific CpG induced higher secondary immune response than that of the pcDNA3-VP2C1 vector.Conclusion These data indicate that the pcDNAK-VP2C2 vector containing canine-specific CpG is highly antigenic and could be used for further studies for development of gene vaccines against canine parvovirus disease.