PPV VP2基因与PCV2 ORF2不同抗原优势区重组真核表达质粒的构建及其免疫效果研究
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摘要
为了研究猪细小病毒VP2基因和猪圆环病毒2型ORF2基因的不同抗原优势区重组真核表达质粒的免疫原性和在免疫动物体内的分布及安全性,分别以PPV SC-1株和PCV2 SC株为模板,扩增了PPV VP2基因和PCV2 ORF2上不同抗原优势区基因A、B、C(A:117～131aa、B:157～183aa、C:165～200aa)。将A、B、C抗原基因分别与PPV VP2基因串联,并插入pCI-neo真核表达载体,构建了能同时表达PPV VP2蛋白和PCV2 Cap蛋白的重组质粒pCI-A-VP2、pCI-B-VP2、pCI-C-VP2。将各重组真核表达质粒转染Vero细胞,用间接免疫荧光试验检测表达产物的免疫学活性；并以小鼠为动物模型,分别将猪细小病毒灭活疫苗、猪圆环病毒亚单位疫苗、pCI-neo空载体和3种重组真核表达质粒pCI-A-VP2、pCI-B-VP2、pCI-C-VP2通过肌注进行免疫,相同剂量免疫2次,间隔2周。采用MTT比色法、流式细胞术和ELISA法检测了免疫小鼠脾脏淋巴细胞的转化功能,外周血中CD4+和CD8+淋巴细胞比例,PCV2和PPV IgG抗体效价。并且为了研究各重组质粒在免疫动物体内的分布和安全性,采集了不同时期免疫小鼠的心、肺、肝、肾及肌肉组织进行基因组DNA的抽提和PCR扩增。结果显示,3种重组真核表达质粒转染Vero细胞48h后,间接免疫荧光试验都能检测到较强的免疫荧光；3种重组质粒免疫小鼠后第7天起,免疫小鼠脾脏淋巴细胞的增殖活性,外周血CD4+、CD8+T淋巴细胞比例和抗PCV2、PPV的IgG抗体效价都显著(P<0.05)或极显著(P<0.01)高于空载体对照组,且免疫效率pCI-C-VP2组最高,pCI-A-VP2组最低；重组质粒免疫小鼠后7-49d在各器官具有广泛的分布,第56d开始只能在心脏及肌肉组织扩增出目的基因片段,其它组织PCR结果全为阴性。而7-56d采集的不同组织样品纯化基因组DNA的PCR实验结果都为阴性。证实所构建的重组真核表达质粒均能在细胞中正常表达；能够诱导免疫小鼠产生良好的细胞免疫和体液免疫反应；对免疫小鼠具有良好的安全性且在各器官具有广泛的分布。该研究结果为PCV2 ORF2基因上抗原优势区域的选择和PPV/PCV2二联疫苗的研究提供了实验依据。
To study the immune efficacies、distributions and securities in animal bodies of recombinant eukaryotic expression plasmids with PPV VP2 gene and PCV2 ORF2 different antigen predominance regionals.VP2 gene was amplified from PPV SC-1 strain,and different antigen genes A、B、C(A:117～131aa、B:157～183aa、C:165～200aa) were amplified from PCV2 SC strain, respectively.The A、B、C genes and PPV VP2 gene were compounded in series and cloned into pCI-neo vector to construct the recombinant plasmids pCI-A-VP2、pCI-B-VP2、pCI-C-VP2.The recombinant plasmids were transfected into Vero cells to study the immune activities by immuno-fluorescent antibody technique. Healthy mice were inoculated with Porcine Parvovirus vaccine and Porcine Circovirus subunit vaccine, pCI-neo DNA and three recombinant plasmids for two times, the immune efficacies were detected by the MTT assay、FCM and ELISA. And the distributions、securities were inspected by PCR amplification.
The results showed that the recombinant proteins were able to express nomally, fluorescence were observed in the cells transfected with pCI-A-VP2、pCI-B-VP2、pCI-C-VP2,and their immune efficacies were significantly higer than pCI-neo,and pCI-C-VP2 was of the higgest immune efficacies in the three recombinant plasmids. The PCR amplification results proved that the distributions were broad and the securities in animal bodies were good.These results provided reference datas for the bivalent vaccine research of PPV and PCV2.
To study the immune efficacies、distributions and securities in animal bodies of recombinant eukaryotic expression plasmids with PPV VP2 gene and PCV2 ORF2 different antigen predominance regionals.VP2 gene was amplified from PPV SC-1 strain,and different antigen genes A、B、C(A:117～131aa、B:157～183aa、C:165～200aa) were amplified from PCV2 SC strain, respectively.The A、B、C genes and PPV VP2 gene were compounded in series and cloned into pCI-neo vector to construct the recombinant plasmids pCI-A-VP2、pCI-B-VP2、pCI-C-VP2.The recombinant plasmids were transfected into Vero cells to study the immune activities by immuno-fluorescent antibody technique. Healthy mice were inoculated with Porcine Parvovirus vaccine and Porcine Circovirus subunit vaccine, pCI-neo DNA and three recombinant plasmids for two times, the immune efficacies were detected by the MTT assay、FCM and ELISA. And the distributions、securities were inspected by PCR amplification.
The results showed that the recombinant proteins were able to express nomally, fluorescence were observed in the cells transfected with pCI-A-VP2、pCI-B-VP2、pCI-C-VP2,and their immune efficacies were significantly higer than pCI-neo,and pCI-C-VP2 was of the higgest immune efficacies in the three recombinant plasmids. The PCR amplification results proved that the distributions were broad and the securities in animal bodies were good.These results provided reference datas for the bivalent vaccine research of PPV and PCV2.
引文
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[23]崔保安,魏战勇,王学斌.IL-2与猪细小病毒VP2基因双表达载体的构建及免疫原性的研究[J].生物工程学报,2006.22(3)：425-430.
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