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

【目的】构建猪繁殖与呼吸综合征病毒(PRRSV)GP5基因C3d-P28分子佐剂重组质粒,探明C3d-P28分子佐剂的免疫增强效果。【方法】用大肠杆菌疫苗对健康猪进行免疫激活,提取猪肝脏总RNA,RT-PCR克隆C3d-P28并连接至pUC19载体,构建pUC19-P28.n(2、4、6)串联体,然后分别将P28.n(2、4、6)连接至真核表达载体pcDNA3.1构建成pcDNA3.1-P28.n(2、4、6)。RT-PCR扩增PRRSV GP5基因,分别定向克隆至pcDNA3.1-P28.n(2、4、6)中,构建pcDNA3.1-GP5-P28.n(2、4、6)重组质粒;提取重组质粒进行双酶切、电泳,同时用重组质粒转染Marc 145细胞,经间接免疫荧光进行蛋白表达检测,并用重组质粒免疫小鼠,通过ELISA试剂盒检测小鼠体内抗体、IL-4和IFN-γ的水平,检验重组质粒的免疫效果。【结果】从猪肝脏中克隆了C3d cDNA,构建了pcDNA3.1-P28.n(2、4、6)串联体,并将PRRSV GP5基因定向克隆至真核表达载体pcDNA3.1-P28.n(2、4、6)中,构建了PRRSV GP5重组质粒(pcDNA3.1-GP5-P28.2、pcDNA3.1-GP5-P28.4、pcDNA3.1-GP5-P28.6);通过检测小鼠血清中抗体、IL-4和IFN-γ的结果显示,抗体效价、IL-4和IFN-γ含量均比对照组高,且以pcDNA3.1-GP5-P28.6组的效果最好,与空白对照及PCDNA3.1-GP5组比较差异均显著(P<0.05)。【结论】构建了猪补体C3d-P28分子佐剂PRRSV GP5重组质粒,该质粒可刺激机体产生较高的体液免疫和细胞免疫水平,补体C3d-P28分子佐剂能显著增强PRRSV GP5基因的免疫效果。

【 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 BamHI 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 of pcDNA3.1-p28.n plasmid,and the DNA vaccines containing GP5 gene with C3d-p28 as molecular adjuvant were successfully constructed.All 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 Mark145 cells tranfected by recombinant plasmids.Furthermore,seven groups of mice were injected with these recombinant plasmids.GP5-specific ELISA antibody,IFN-γlevel 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.