中国农业科学2011，Vol.44Issue(12)：2582-2588,7.DOI:10.3864/j.issn.0578-1752.2011.12.022
猪C3d分子佐剂PRRSV GP5基因核酸疫苗的构建及免疫效果的研究
Construction and Immunogenicity of DNA Vaccines Containing GP5 Gene Against PRRSV with Porcine C3d as Molecular Adjuvant
摘要
Abstract
[Objective] DNA vaccines containing PRRSV GP5 gene with porcine C3d-p28 were constructed and the enhancing effect of molecular adjuvant C3d-p28 was proved. [Method] Pigs were injected with the oil emulsion of Escherichia coli inactivated vaccine in order to induce immune activation. After cloning the C3d cDNA of pig by using liver as the source of mRNA, a pair of primers was designed to sub clone the P28 gene to the pUC19 plasmid. Several tandems of p28 were constructed in the pUC19 plasmid using a pair of isoschizomers BarnHO and Bgl Ⅱ. The genes of p28.n, which were digested from pUC19-p28.n, were cloned into pcDNA3.1 (+) plasmid. The GP5 gene of PRRSV was cloned by RT-PCR and inserted into the upstream ofpcDNA3.1-p28.n plasmid, and the DNA vaccines containing GP5 gene with C3d-p28 as molecular adjuvant were successfully constructed. AH recombinant plasmids were verified by PCR, restriction endonuclease digestion and DNA sequencing. In addition, indirect immunofluorescence assay (IFA) could be found within the cells after Mark 145 cells tranfected by recombinant plasmids. Furthermore, seven groups of mice were injected with these recombinant plasmids. GP5-specific ELISA antibody, IFN- y level and IL-4 level in the sera were detected. [ Result ] It was showed that the C3d cDNA of pig was cloned and several tandems of P28 and pcDNA3.1 (+)-GP5-C3d-p28.n were successfully constructed. Serological analysis showed that BALB/c mice that vaccinated with DNA vaccine expressing fusions of pcDNA3.1-GP5-p28 (two, four or six repeats) elicited a higher level of humoral response compared with mice that vaccinated with DNA vaccine expressing only the pcDNA3.1 vector and pcDNA3.1-GP5 group (P <0.05). The increase in the immune response elicited by six copies of p28 was the highest. [Conclusion] The DNA vaccine containing PRRSV GP5 gene with porcine C3d-p28 has been successfully constructed, which could elicit humoral and cellular immune responses. The complement C3d-p28 can significantly enhance the nucleic acid vaccine immune effect as molecular adjuvant.关键词
PRRSV/C3d-p28佐剂/GP5基因疫苗/免疫效果Key words
PRRSV/C3d-p28 molecular adjuvant/GP5 DNA vaccine/effect of immunity引用本文复制引用
夏庆祥,张德庆,吴家强,牛星,王小龙,牛钟相..猪C3d分子佐剂PRRSV GP5基因核酸疫苗的构建及免疫效果的研究[J].中国农业科学,2011,44(12):2582-2588,7.基金项目
山东省自然科学基金(Y2007D47) （Y2007D47）
