PPV VP2基因与PCV2 ORF2不同抗原表位重组真核表达载体的构建及其免疫原性

Construction and immune efficacy of eukaryotic expression vectors of PPV VP2 gene with different antigen epitopes of PCV2 ORF2 gene

为了研究PCV2ORF2的不同抗原表位重组PPV VP2基因真核表达质粒的体外表达情况与免疫原性,分别以PCV2SC株和PPV SC-1株为模板,扩增PCV2ORF2的抗原表位基因A、B、C(A:117～131aa,B:157～183aa,C:165～200aa)和PPV VP2基因。将A、B、C基因分别与PPV VP2基因串联,并插入到pCI真核表达载体中,构建了能同时表达PPV VP2蛋白和PCV2ORF2抗原表位的重组质粒pCI-A-VP2、pCI-B-VP2、pCI-C-VP2。将重组真核表达质粒转染MDBK细胞,用间接免疫荧光试验检测各个质粒在细胞中的表达情况;并将其免疫小鼠,采用MTT比色法、流式细胞术和ELISA法检测免疫效果。结果显示,3种重组表达质粒转染细胞48h后都能检测到较强的免疫荧光;免疫小鼠后14～42d诱导的T淋巴细胞转化效率、CD4+/CD8+细胞比值以及PPV和PCV2抗体均显著高于对照组,且pCI-B-VP2免疫效率高于其他组。该结果表明PCV2ORF2上157～183aa抗原表位在3个抗原表位中抗原性最强,可作为PCV2与其他病毒二联疫苗研究的主要抗原表位。

To investigate the immune efficacy of recombinant plasmids of PPV VP2 with different antigen epitopes of PCV20RF2,the antigen epitope of A（117-131 aa）,B（157-183 aa）,C（165-200 aa） and VP2 gene were obtained by PCR from PCV2 SC strain and PPV SC-1 strain respectively, and three recombinant plasmids including pCI -A-VP2, pCI-B-VP2,pCI-C-VP2 were constructed separately. Three recombinant plasmids were transferred into MDBK cells respectively,and examined by indirect immunofluorescence technology. Female mice were inoculated with the recombinant plasmids respectively,and the immune efficacy was detected by the MTT assay, FCM and ELISA. Fluorescence was ob- served in the cells after transfection with pCI -A-VP2, pCI-B-VP2, pCI-C-VP2. The immune efficacy of three recombinant plasmids was significantly higher than the negative control group and pCI-B-VP2 was the highest among the three plasmids. It indicated that antigen epitopes B of PCV20RF2 can be used to study the two joint vaccines with other virus.