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| 山羊流产嗜衣原体OmpA基因重组质粒构建及对小鼠免疫效果研究 | |
| 引用本文： | 龙冲冲,邓位喜,毛以智,曾贵英,伍波涛,熊朝丽,徐茂文,夏鹏,文明.山羊流产嗜衣原体OmpA基因重组质粒构建及对小鼠免疫效果研究[J].中国畜牧兽医,2016,43(11):2886-2891. |
| 作者姓名： | 龙冲冲 邓位喜 毛以智 曾贵英 伍波涛 熊朝丽 徐茂文 夏鹏 文明 |
| 作者单位： | 1. 遵义市农业委员会, 遵义 563000; 2. 贵州大学动物科学学院, 贵阳 550025 |
| 基金项目： | 贵州省省市科技合作专项资金项目（省市重大[2013]4号）；贵州省科技创新人才团队（黔科合人才团队[2015]4016号） |
| 摘 要： | 为探讨山羊流产嗜衣原体重组真核质粒进入临床试验的可行性，本试验用PCR方法扩增出山羊流产嗜衣原体OmpA基因，克隆至真核表达载体pcDNA3.1（+）中，经PCR和双酶切鉴定后，将重组质粒pcDNA3.1-OmpA转染至PK-15细胞，观察目的基因表达情况，并将制备的大肠杆菌基因组单链DNA作为分子佐剂与重组质粒共同免疫小鼠，检测重组质粒在小鼠体内分布情况及血清抗体水平。结果表明，经PCR、双酶切和测序鉴定表明成功构建重组质粒pcDNA3.1-OmpA；转染PK-15细胞后，荧光抗体试验结果证实重组质粒pcDNA3.1-OmpA得到有效表达。首免后14 d，重组质粒加分子佐剂组的抗体效价显著高于重组质粒组和灭活疫苗组（P<0.05）；随着免疫次数增加和时间推移，免疫小鼠抗体水平均呈现上升趋势，至35 d时达到最高峰，此后抗体滴度逐渐下降，但仍维持较高水平。首免后21 d，在小鼠心脏、肝脏、脾脏、肾脏、肺脏、脑、空肠和腿肌中均可检测到质粒的分布，此后逐渐消失，49 d在所检组织中均未检测到重组质粒的存在。表明试验成功构建了基于OmpA基因的山羊流产嗜衣原体重组真核质粒，且加入分子佐剂后可诱导小鼠产生较高水平的血清抗体。 |
| 关 键 词： | 流产嗜衣原体 OmpA基因 分子佐剂 免疫效果 |
| 收稿时间： | 2016-04-27 |
Construction of Recombined Plasmid with OmpA Gene of Goat Chlamydophila abortus and Immunogenicity Effect in Mice |
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| LONG Chong-chong,DENG Wei-xi,MAO Yi-zhi,ZENG Gui-ying,WU Bo-tao,XIONG Chao-li,XU Mao-wen,XIA Peng,WEN Ming.Construction of Recombined Plasmid with OmpA Gene of Goat Chlamydophila abortus and Immunogenicity Effect in Mice[J].China Animal Husbandry & Veterinary Medicine,2016,43(11):2886-2891. | |
| Authors: | LONG Chong-chong DENG Wei-xi MAO Yi-zhi ZENG Gui-ying WU Bo-tao XIONG Chao-li XU Mao-wen XIA Peng WEN Ming |
| Institution: | 1. Agricultural Commission of Zunyi Municipal, Zunyi 563000, China; 2. College of Animal Science, Guizhou University, Guiyang 550025, China |
| Abstract: | To explore the feasibility of entering clinical trials of the goat Chlamydophila abortus eukaryotic plasmids,Chlamydophila abortus OmpA gene was amplified by PCR and cloned into the eukaryotic expressing plasmid pcDNA3.1(+）to construction the recombinant vetor.After identification by PCR and restriction enzyme digestion,this vector was transfected into PK-15 cells and its expression were observed by fluorescent antibody test,the distribution of serum antibodies and plasmid were detected in mice after the immunization of the recombinant vector and molecular adjuvant which was single-stranded DNA of E.coli bacterial genome.The results showed that the recombinant plasmid pcDNA3.1-OmpA was successfully constructed after detecting by PCR,enzyme digestion and sequencing.The OmpA gene was effectively expressed in PK-15 cells.The anti-OmpA antibody levels of the pcDNA3.1-OmpA with molecular adjuvant group was significantly higher than that in pcDNA3.1-OmpA group and the inactivated vaccine group at 14 d after immunization（P<0.05).This levels showed an upward trend following numbers of immunization and times.The highest levels was at 35 d after immunization.Then the antibody titers were gradually decreased which still maintain a higher antibody levels than before.The pcDNA3.1-OmpA could be detected in the heart,liver,spleen,kidney,lung,brain,jejunum and leg muscle of mice on 21 d after immunization,and couldn't be detected in any organs at 49 d after immunization.The results above indicated that the the recombinant vetor based on OmpA gene of Chlamydophila abortus was successfully constructed in this experiment,after joining the molecular adjuvant could induce to a higher level of serum antibodies in mice. |
| Keywords: | Chlamydophila abortus OmpA gene molecular adjuvant immune effect |
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