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| 表达H5亚型禽流感同义(Consensus)HA蛋白的重组质粒构建及体外表达 | |
| 引用本文： | 常晓飞,赵双成,田石,柳金雄,王靖飞,曾显营,胡永浩,姜永萍,陈化兰.表达H5亚型禽流感同义(Consensus)HA蛋白的重组质粒构建及体外表达[J].中国预防兽医学报,2012,34(7):510-513. |
| 作者姓名： | 常晓飞 赵双成 田石 柳金雄 王靖飞 曾显营 胡永浩 姜永萍 陈化兰 |
| 作者单位： | 1. 甘肃农业大学动物医学院,甘肃兰州730007;中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/农业部动物流感重点开放实验室,黑龙江哈尔滨150001 2. 中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/农业部动物流感重点开放实验室,黑龙江哈尔滨,150001 3. 中国农业科学院哈尔滨兽医研究所疫病诊断与技术服务中心,黑龙江哈尔滨,150001 4. 甘肃农业大学动物医学院,甘肃兰州,730007 |
| 基金项目： | 哈尔滨市科技攻关计划项目 |
| 摘 要： | 为研究禽流感病毒(AIV)H5亚型同义Consensus HA(Cons-HA5)基因的重组表达质粒在体外的表达情况,本研究通过对从NCBI流感数据库中获得的5000条H5亚型AIV的HA蛋白的序列比对、分析获得一条同义HA蛋白,将其相对应的核苷酸序列进行鸡体偏嗜性密码子优化,人工合成Cons-HA5基因并克隆于真核表达载体pCAGGS中构建重组表达质粒pCACons-HA5。将重组质粒转染293-T细胞,在激光共聚焦显微镜下观察转染后不同时间HA蛋白的表达情况,同时在转染后24 h、48 h后分别进行表达蛋白的western blot检测。结果表明,pCACons-HA5转染293-T细胞后,其表达的HA蛋白先分布于细胞质中,而后转移至细胞膜表面;westernblot鉴定结果表明,Cons-HA5重组蛋白可以与AIV多克隆血清反应分子量约为70 ku。该重组质粒的构建将为进一步Cons-HA5核酸免疫对H5亚型不同抗原群AIV的交叉保护免疫效力研究奠定基础。 |
| 关 键 词： | H5亚型禽流感病毒 同义HA基因 表达 |
Construction and in vitro expression of consensus-hemagglutinin-based recombinant plasmid of H5 subtype avian influenza virus |
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| CHANG Xiao-fei , ZHAO Shuang-cheng , TIAN Shi , LIU Jin-xiong , WANG Jing-fei , ZENG xian-ying , HU Yong-hao , JIANG Yong-ping , CHEN Hua-lan.Construction and in vitro expression of consensus-hemagglutinin-based recombinant plasmid of H5 subtype avian influenza virus[J].Chinese Journal of Preventive Veterinary Medicine,2012,34(7):510-513. | |
| Authors: | CHANG Xiao-fei ZHAO Shuang-cheng TIAN Shi LIU Jin-xiong WANG Jing-fei ZENG xian-ying HU Yong-hao JIANG Yong-ping CHEN Hua-lan |
| Institution: | 1.College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730007,China;2.State Key Laboratory of Veterinary Biotechnology,Animal Influenza Laboratory of the Ministry of Agricultural,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150001,China;3.Animal Disease Diagnostic Center,Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences,Harbin 150001,China) |
| Abstract: | To expression a consensus HA(Cons-HA5) gene of H5 subtype avian influenza virus(AIV) and localize the recombinant protein in transfected cells,a Cons-HA5 gene was designed by clustering 5,000 HA protein sequence of H5 subtype AIV available in GenBank and synthesized based on chicken-bisaed codon-optimization.The Cons-HA5 gene was cloned into the eukaryotic expression vector of pCAGGS to construct the recombinant plasmid pCACons-HA5.The pCACons-HA5 was transfected into 293-T cells for expression and identification.The result showed that the localization of expressed Cons-HA5 was initially found in the cytoplasm and then transported to the cell surface at 4 hours post transfection.Western blot result indicated that the molecular weight of Cons-HA5 was about 70 ku.These results provided a base for further study of the Cons-HA5 based DNA vaccine for protection against H5 subtype AIV strains in chickens. |
| Keywords: | H5 subtype avian influenzas virus Consensus HA gene expression |
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