[目的]为了提高犬传染性肝炎Hexon蛋白基因在大肠杆菌中的表达量,研究Hexon基因蛋白的最佳表达条件和表达蛋白的活性.[方法]在大肠杆菌菌株BL21(DE3)培养过程中,选择诱导剂(IPTG)的最佳加入时间、诱导剂(IFTG)浓度、诱导时间以及不同温度条件观察对Hexon融合蛋白表达的影响,以确定最佳表达条件,并用Westem-blot检测重组蛋白的生物活性.[结果]大肠杆菌BL21(DE3)在37℃培养2.25h后,添加终浓度为2.0 mmol/L的IPTG,27℃培养8 h,Hexon重组蛋白表达量最高(1.67mg/mL),所表达的重组蛋白能与犬传染性肝炎阳性血清相结合.[结论]在高浓度IPTG诱导和冷培养条件下,Hexon蛋白基因的表达量最高,表达出的重组蛋白具有很好的反应原性,为进一步研究六邻体蛋白的作用机理及亚单位疫苗的研究提供了实验基础.%[Objective]In order to improve the expression level of Hexon gene of canine Infectious Hepatitis virus( CIHV)in Escherichia coli , the optimization of expression conditions of Hexon gene and the biological activity of expressed Hexon fusion protein were studied in this paper. [Method] The influence of various expressing conditions, including the time of adding IPTG , IPTG concentration, induction time and temperature on expression of Hexon gene were observed to decide the optimal conditions . The biological activity of expressed fusion protein was detected by Westem - blot . [ Result ] The expressed recombinant protein Hexon reached the highest level ( 1 . 67 mg/mL) in transformed bacterium strain Escherichia coli BL21 ( DE3) after pre - culture for 2. 25 h ( OD600 = 0.5) ,adding IPTG to make final concentration to 2 mmol/L and continuing to culture 8 h at 27 ％ . and could be combined with positive serum of CIHV. [ Conclusion]In the condition of high concentration of IPTC and cold temperature , the expression level of recombinant protein Hexon was the highest . The expressed recombinant protein Hexon had high reactivity to positive serum of CIHV. It provides a good foundation to further study on the active mechanism of Hexon protein and subunit vaccine against CIHV.
展开▼
