| 引用本文： |
-
刘学东,包振民,王志亮.禽流感病毒H5N1亚型基因工程疫苗设计、表达制备及动物实验研究[J].同济大学学报（医学版）,2011,32(6):1-8. [点击复制]
- LIU Xue-dong,BAO Zhen-min,WANG Zhi-liang.Preparation of avian influenza virus H5N1 genetic engineering vaccine and animal study[J].同济大学学报（医学版）,2011,32(6):1-8. [点击复制]
|
|
| 摘要: |
| 目的 设计并获得一种安全、高效,并能对以H5N1亚型为主的多种禽流感病毒亚型产生保护的复合基因工程疫苗。方法 利用分子生物学和分子免疫学方法对国内以及周边国家流行的禽流感毒株进行全面分析,利用生物信息学技术合理设计亚单位加表位的全新复合结构疫苗的氨基酸序列;利用大肠杆菌密码子优化技术根据疫苗氨基酸序列获得基因序列;通过全基因合成的方法获得该疫苗的基因,并通过大肠杆菌表达系统对该疫苗进行表达;目的蛋白经过包涵体洗涤和色谱纯化后复性;通过ELISA评价其抗原特异性,并乳化制备成禽流感蛋白疫苗,通过小鼠体内免疫试验检测其免疫效力。结果 设计了包含通用性的辅助性T细胞表位、B细胞表位以及CTL表位的串接疫苗结构;全基因合成获得了该复合疫苗的基因序列;该基因在大肠杆菌表达系统中得到了高效表达,目的蛋白表达量约占菌体总蛋白30%;经过粗纯和精纯后的目的蛋白纯度达到95.5%,复性后获得可溶性蛋白溶液,浓度可达2.4 mg/mL。结论 通过小鼠实验,验证了该H5N1亚型基因工程疫苗的抗原性,证实该基因工程疫苗在免疫小鼠体内激发体液免疫的同时调动了细胞免疫。小鼠免疫试验证明,该疫苗可以在小鼠体内有效诱发免疫应答。 |
| 关键词: 禽流感 疫苗 原核表达 蛋白质纯化 蛋白质复性 H5N1 免疫效力 小鼠 |
| DOI：10.3969/j.issn1008-0392.2011.06.001 |
|
| 基金项目: |
|
| Preparation of avian influenza virus H5N1 genetic engineering vaccine and animal study |
| LIU Xue-dong1,2,BAO Zhen-min2,WANG Zhi-liang3 |
| (1.Genetics and Breeding Laboratory,Ocean University of China,Qingdao 266033,China; 2.Dept.of Respiratory Medicine,Qingdao Municipal Hospital,Qingdao 266011,China;3.China Animal Health and Epidemiology Center,Qingdao 266008,China) |
| Abstract: |
| Objective To design and prepare a genetic engineering vaccine against avian influenza virus H5NI.Methods The composite amino acid sequences plus epitope subunit of vaccine were designed based on the analysis of prevalent avian influenza H5NI strains and bioinformatics.The amino acid sequence was obtained with E.coli codon optimization technique;the vaccine gene was synthesized with the whole gene synthesis method and expressed with E.coli expression system.The protein inclusion bodies were purified by renaturation chromatography after washing;the antigen specificity was measured by ELISA.The emulsified protein vaccine of avian influenza prepared was evaluated by immunization test in mice for immune effect.Results The target protein expressed in E. coli accounted for approximately 30%of the total bacterial protein;the purity of protein was 95.5% after purification and the concentration of soluble protein was up to 2.4 mg/ml.Immune tests proved that the vaccine effectively induced immune responses in mice.Conclusion The engineering vaccine against avian influenza virus H5N1 has been successfully prepared which can induce immune response in mice. |
| Key words: Avian influenza vaccine prokaryotic expression protein purification protein refolding H5N1 immune effect mouse |
