| 不同温度、时间、IPTG和菌种浓度对丁肝抗原蛋白表达量的影响 |
| 投稿时间：2011-11-21 修订日期：2011-11-24 点此下载全文 |
| 引用本文：丁军颖,伊瑶,卢学新,苏秋东,田瑞光,邱丰,毕胜利.不同温度、时间、IPTG和菌种浓度对丁肝抗原蛋白表达量的影响[J].医学研究杂志,2012,41(5):31-34 |
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| 基金项目:国家科技重大专项基金资助项目（2008ZX10201） |
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| 中文摘要:目的确定以大肠杆菌表达丁肝抗原的最佳条件，为规模生产以求研发丁肝ELISA诊断试剂奠定基础。方法在不同温度、时间、IPTG和菌种浓度条件下，分别进行蛋白表达，取等体积样本进行SDS-PAGE电泳，经Image Lab软件分析，比较目的蛋白表达量，确定最佳表达条件。结果在25℃、29℃、33℃和37℃条件下，蛋白表达量无统计意义的差别；在添加IPTG后的1、2、3、4、5和6h，表达量无统计意义的差别；在以0.25、0.5、1、2和4mmol/L IPTG诱导时，表达量无统计意义的差别。当添加诱导剂前，菌种的OD600值分别为1.236、0.772、0.542和0.384，目的蛋白表达量有显著意义的差别。以OD600值为 1.236和0772时，表达量最高，两者之间无统计意义的差别；OD600值为0.542 时，表达量次之；OD600值为 0.384时，表达量最低。结论温度、表达时间和IPTG浓度在一定范围内对丁肝抗原蛋白表达量无影响；菌种的浓度是影响蛋白表达量的决定性因素；当菌种浓度在OD600值约为0.7~1.2时，以0.25mmol/L IPTG在37℃，诱导表达1h为最佳表达条件。该条件下，目的蛋白表达量占菌体总蛋白的41.5%。 |
| 中文关键词:丁肝 基因工程 抗原 表达 |
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| Effect of Different Temperature，Time and Different Concentration of IPTG and Strains on HDVAg Expression |
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| Abstract:ObjectiveTo determine the optimum HDAg expression condition in E. coli and to prepare for the study of HDV diagnosis reagent in large scale. MethodsProtein was expressed in different conditions, including temperature, time, the concentration of IPTG and bacterial strain. The SDS-PAGE was made，then the target protein yields were compared with Image Lab Software. The optimum protein expression parameters were determined.ResultsIn different temperature or time, the target protein yields had no statistic significance. In different concentration of IPTG, the protein yields had no statistic significance, too. Which OD600 was 1.236、0.772、0.542 or 0.384, the bacterial strain was induced by IPTG separately. The protein yields of these groups had statistic significance. The highest protein yield was achieved when the OD600 was 0.772 and 1.236, the lower was achieved when the OD600 was 0.542, while the lowest was got when the OD600 was 0.384 respectively. ConclusionTemperature, time, the concentration of IPTG have no effect on the HDAg protein yield. The concentration of bacterial strain plays an important role in the protein expression. In the optimum expression conditions, the target protein takes 45% proportion of the total bacterial protein. |
| keywords:HDAg Genetic engineering Antigen Express |
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