| 白细胞介素-12对犬细小病毒VP2 DNA疫苗的免疫增强作用 |
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| 引用本文： | 潘素敏,李秀锦,仲飞,柳新保,韩冬梅,王幸兴,潘宏丽,王微.白细胞介素-12对犬细小病毒VP2 DNA疫苗的免疫增强作用[J].兽医大学学报,2012(2):196-201. |
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| 作者姓名： | 潘素敏 李秀锦 仲飞 柳新保 韩冬梅 王幸兴 潘宏丽 王微 |
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| 作者单位： | [1]河北农业大学动物科技学院基础兽医部,河北保定071001 [2]燕山大学环境与化学工程学院生物工程系,河北秦皇岛066004 [3]河北科技师范学院动物科技学院基础兽医部,河北秦皇岛066000 [4]广东大台农饲料有限公司,广东广州510550 |
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| 基金项目： | 国家自然科学基金资助项目（30771586）; 河北省自然科学基金资助项目（C2008000244）; 河北省人事厅留学人员科技活动择优资助项目（20080808）（感谢中国农业大学生物学院刘维全教授给予实验材料和技术上的支持） |
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| 摘 要： | 犬细小病毒编码的VP2蛋白是该病毒重要的结构蛋白和抗原蛋白。利用VP2基因制备的DNA疫苗能够刺激机体产生免疫应答反应。为进一步提高VP2DNA疫苗的免疫应答水平,本研究在小鼠体内尝试了利用白细胞介素12（IL-12）基因表达载体提高VP2DNA疫苗的免疫应答水平。首先采用RT-PCR方法从小鼠脾淋巴细胞中分别扩增IL-12大亚基（P40）和小亚基（P35）cDNA基因;然后在真核表达载体pcDNA3.1A上通过引入内部核糖体进入位点（IRES）序列,分别将P40基因和P35基因插入到IRES序列的上下游,构建成IL-12（P40和P35双亚基）基因表达载体,pcDNA-P40-IRES-P35。将上述表达载体与本室构建的VP2表达载体通过磷酸钙方法转染HEK 293T细胞进行瞬时表达,以确定构建的表达载体能否介导相应基因在真核细胞中进行分泌表达。然后用VP2载体单免疫和VP2载体和IL-12载体共免疫方法对小鼠进行免疫（用pcDNA3.1A作为对照）。免疫后在特定时间通过ELISA方法检测小鼠血清抗VP2蛋白的抗体水平,并通过淋巴细胞增殖实验检测免疫后35d小鼠脾脏淋巴细胞增殖反应。结果表明,扩增的小鼠IL-12P40和P35亚基基因与GenBank的参考序列基本一致。Western-blot检测结果表明,重组IL-12和VP2均能够在HEK293T细胞中进行分泌性表达。ELISA检测结果表明利用IL-2载体与VP2载体共免疫小鼠,其血清中抗VP2的抗体水平明显高于VP2载体单免疫组（P〈0.01）,抗体水平在第35天高达1：5120。淋巴细胞增殖试验结果表明,免疫小鼠的淋巴细胞刺激指数均明显高于对照组（P〈0.01）,VP2载体与IL2载体共免疫组的刺激指数明显高于VP2载体单免疫组（P〈0.05）。由此可见,在小鼠体内,IL-12基因表达载体可明显提高CPV VP2基因疫苗的免疫应答水平。
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| 关 键 词： | IL-12 细小病毒 VP2基因 DNA疫苗 |
Enhancement of canine parvovirus VP2 DNA vaccine potency by coadministration with interleukin-12 gene expression vector |
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| Authors: | PAN Su-min LI Xiu-jin ZHONG Fei LIU Xin-bao HAN Dong-mei WANG Xing-xing PAN Hong-li WANG Wei |
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| Institution: | 1.College of Veterinary Medicine,Hebei Agricultural University,Baoding,Hebei 071001,China;2.College of Environmental and Chemical Engineering,Yanshan University,Qinhuangdao,Hebei 066004,China;3.College of Animal Science,Hebei Normal University of Science and Technology,Qinhuangdao,Hebei 066000,China;4.Guanzhou Datainong Feed Limited Company,Guangzhou 510550,China) |
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| Abstract: | Canine parvovirus（CPV） VP2 protein is a major structural and antigenic protein.It has been report that VP2 gene vaccine can stimulate the immune responses against CPV in animals.To further enhance VP2 DNA vaccine immune response,we tested to use interleukin 12（IL-12） gene to improve VP2 DNA vaccine potency in mice.First,mouse IL-12（mIL-12） two subunit cDNA（P40 and P35） were amplified from mouse spleen cells by RT-PCR and then subcloned into the upstream and downstream of an internal ribosomal entry site（IRES） in pcDNA3.1A-IRSE vector to construct mIL-12 double-gene eukaryotic expression vector.The mIL-12 vector and VP2 vector prepared previously were tansfected into HEK293T cells by calcium phosphate mediation to determine whether recombinant mIL-12 and VP2 protein can be expressed and secreted in eukaryotic cells.The mice were immunized with VP2 gene and VP2 gener plus mIL-12 gene,respectively.The antibody titers in serum of the mice were measured using ELISA.The spleen lymphocyte proliferation activity was analyzed by 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenylte-trazolium bromide（MTT）assay.The results showed that the sequence of mIL-12 gene amplified in this study was basically identical with the published sequence.The recombinant mIL-12 protein and VP2 protein could be expressed and secreted in HEK293T cells.The mice immunized with either VP2 gene or VP2 gene plus mIL-12 gene could generate the specific antibody against VP2 protein.Howere the antibody titer immunized with VP2 gene plua mIL-12gene was significantly higher than that of VP2 gene only（P0.01）,reached 1：5120 at 35d post immunization.For lymphocyte proliferation aasay,lymphocyte stimulation index of the mice immunized with VP2 gene plus IL-12 gene was significantly higher（P0.05） that of with VP2 gene.Thus mIL-12 gene could enhance the immune response of CPV VP2 gene vaccine in mice. |
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| Keywords: | IL-12 canine parvovirus VP2 gene DNA vaccine |
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