| 兔出血症病毒VP60基因真核表达质粒的构建及初步评价 |
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| 引用本文： | 原冬伟,刘家森,郭东春,司昌德,姜骞,林欢,穆亚芳,刘行波,曲连东.兔出血症病毒VP60基因真核表达质粒的构建及初步评价[J].中国兽医学报,2011,31(11). |
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| 作者姓名： | 原冬伟 刘家森 郭东春 司昌德 姜骞 林欢 穆亚芳 刘行波 曲连东 |
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| 作者单位： | 中国农业科学院哈尔滨兽医研究所实验动物中心兽医生物技术国家重点实验室,黑龙江哈尔滨,150001 |
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| 基金项目： | 兽医生物技术国家重点实验室开放基金资助项目(SKLVBD201002) |
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| 摘 要： | 利用pMD18-T-VP60质粒（包含RHDVTP株VP60基因）PCR扩增并酶切回收VP60基因片段，插入到真核表达载体pcDNA3．1（＋）中，构建真核表达质粒pcDNA—VP60，对重组质粒进行酶切和PCR鉴定；将重组质粒转染RK13细胞，进行间接免疫荧光和Western-blot检测；将构建好的重组质粒作为核酸疫苗，腿部肌肉注射免疫小鼠，采用间接ELISA法检测抗体水平；采用淋巴细胞增殖试验（MTT法）和流式细胞术（FACS）检测细胞免疫情况；采用PCR方法检测重组质粒在小鼠体内的生物安全性。结果显示，成功构建pcDNA—VP60真核表达质粒，并在RK13细胞中获得表达。重组质粒作为核酸疫苗能够诱导小鼠体内产生特异性抗体而且抗体水平随免疫次数增加而逐渐增高，在免疫后49d时达到峰值；脾淋巴细胞刺激指数和CD4＋、CD8＋淋巴细胞亚群数量百分率明显增高，且与对照组存在明显差异。经PCR鉴定VP60基因未整合到小鼠主要脏器染色体上。结果表明，构建的pcDNA—VP60真核表达质粒能够诱导小鼠产生较强的体液免疫和细胞免疫应答，重组质粒对小鼠是安全的。
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| 关 键 词： | 核酸疫苗 真核表达 VP60 ELISA T淋巴细胞 PCR检测 |
Development of VP60 gene vaccine against RHDV and immunity test in mice |
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| YUAN Dong-wei,LIU Jia-sen,GUO Dong-chun,SI Chang-de,JIANG Qian,LIN Huan,MU Ya-fang,LIU Xing-bo,QU Lian-dong.Development of VP60 gene vaccine against RHDV and immunity test in mice[J].Chinese Journal of Veterinary Science,2011,31(11). |
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| Authors: | YUAN Dong-wei LIU Jia-sen GUO Dong-chun SI Chang-de JIANG Qian LIN Huan MU Ya-fang LIU Xing-bo QU Lian-dong |
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| Institution: | YUAN Dong-wei,LIU Jia-sen,GUO Dong-chun,SI Chang-de,JIANG Qian,LIN Huan,MU Ya-fang,LIU Xing-bo,QU Lian-dong(State Key Laboratory of Veterinary Biotechnology,Experimental Animal Research Center,Harbin Veterinary Research Institite of Chinese Academy of Agricultural Sciences,Harbin 150001,China) |
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| Abstract: | The VP60 gene of RHDV was inserted into the eukaryotic expression vector pcDNA3.1(+) to construct nucleotide vaccine plasmid pcDNA-VP60.The nucleotide vaccine plasmid identified by PCR and digestion was transfected into RK13 cells for the analysis of immunofluorescence and Western-blot.The constructed pcDNA-VP60 was developed into nucleotide vaccine to intramuscularly inject into the mice.The serum were collected to test the antibody level by ELISA.And cell mediated immunity was detected by MTT method and f... |
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| Keywords: | DNA vaccine eukaryotic expression VP60 ELISA T lymphocyte PCR test |
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