Objective Hepatitis Delt a Virus（HDV） antigen is widely used as a capture antigen in ELISAs for the identification of HDV infection; large amounts of recombinant HDV antigen with active antigenicity are required for...Objective Hepatitis Delt a Virus（HDV） antigen is widely used as a capture antigen in ELISAs for the identification of HDV infection; large amounts of recombinant HDV antigen with active antigenicity are required for this purpose. Methods Reconstruct the gene of HDV antigen based on the bias code of Escherichia coli, the recombinant protein expresses by high-density fermentation with fed-batch feeding strategy, and purify by immobilized metal chromatography. The sensitivity and specificity of this antigen detect by ELISA method. Results The expression of HDV antigen can reach 20% of the total cell mass in the soluble form. The recombinant HDV antigen can be conveniently purified（98%） by immobilized metal ion affinity chromatography（IMAC） using the interaction between a His-tag and nickel ions. Production of recombinant HDV antigen can reach 0.5 g/L under conditions of high-density cell fermentation. Applied to the diagnostic ELISA method, the recombinant HDV antigen shows excellent sensitivity（97% for IgM and 100% for IgG） and specificity（100% for IgG and IgM） for the detection of anti-HDV antibodies. Conclusion Expression and purification the recombinant HDV antigen as a candidate protein for application in a diagnostic ELISA for HDV infection. Large-scale production of the protein can be achieved using the high-density fermentation strategy.展开更多
The kinetics of batch and fed-batch cultures of recombinant Escherichia coli producing human-like collagen was investigated. In the batch culture, a kinetic model of a simple growth-association system was concluded wi...The kinetics of batch and fed-batch cultures of recombinant Escherichia coli producing human-like collagen was investigated. In the batch culture, a kinetic model of a simple growth-association system was concluded without consideration of cell endogeneous metabolism. The cell lag time, the maximum specific growth rate and Yx/s were determined as 1.75h, 0.65h^-1 and 0.51g·g^-1, respectively. In the fed-batch culture, different specific growth rates were set at （0.15, 0.2, 0.25h^-1） by the method of pseudo-exponential feeding, and the expressions for the specific rate of substrate consumption, the growth kinetics and the product formation kinetics of each phase were obtained. The result shows that the concentrations of cell and product can reach 77.5g·L^-1 and 10.2g·L^-1 respectively. The modal predictions are in good agreement with the experimental data.展开更多
Several technical parameters were studied during the fermentation of recombinant E.coli for the production of collagen-like biopolymer.The effects of dissolved oxygen as well as glucose concentration on fermentation w...Several technical parameters were studied during the fermentation of recombinant E.coli for the production of collagen-like biopolymer.The effects of dissolved oxygen as well as glucose concentration on fermentation were observed.The OD 600 value could reach 98 when dissolved oxygen was controlled at 50% and glucose around 1%.The production of human-like collagen with a yield of 29.4% was obtained.展开更多
厌氧发酵过程中,物料分层及各层料液黏度、密度等参数是搅拌装置设计的重要依据。该文采用接力试验研究了牛粪中温厌氧发酵过程中各层料液基础物性的变化情况。结果表明:牛粪厌氧发酵过程中,反应器中料液密度自上而下逐渐变大,且各层密...厌氧发酵过程中,物料分层及各层料液黏度、密度等参数是搅拌装置设计的重要依据。该文采用接力试验研究了牛粪中温厌氧发酵过程中各层料液基础物性的变化情况。结果表明:牛粪厌氧发酵过程中,反应器中料液密度自上而下逐渐变大,且各层密度随总固体含量增加而升高,反应过程中各层密度均在发酵第4天达到最大值,料液总固体质量分数为4%、6%、8%时对应的上中下层溶液的密度最大值分别为1.02、1和1.02,1.02、1.03和1.07,1.03、1.03和1.07 g/cm3。受料液分层的影响,反应器中上层和中层料液黏度呈先增大后减小并逐步趋于稳定,下层料液黏度以初始黏度为最大,且不同初始料液总固体含量对黏度变化过程具有显著影响,TS=4%时,上、中层料液黏度分别在第7天、第4天达到最大值11.5和14.7 m Pa·s,下层初始最大黏度为107 m Pa·s;TS=6%、8%时,上、中层料液黏度均在第4天达到最大值,上层料液黏度最大值分别为25.5和63.5 m Pa·s,中层料液黏度最大值分别为15.5和95.5 m Pa·s,下层初始最大黏度分别为135.5和185.5 m Pa·s。随着初始物料总固体含量的增加日产气量也相应增高,产气高峰出现时间相应提前;TS=8%时的累积产气量分别比6%和4%提高了21.4%和8.71%,但产气中甲烷含量增加速率基本相同,并在第10天左右基本趋于稳定。该结果可为厌氧发酵反应器的搅拌装置设计提供参考。展开更多
文摘Objective Hepatitis Delt a Virus（HDV） antigen is widely used as a capture antigen in ELISAs for the identification of HDV infection; large amounts of recombinant HDV antigen with active antigenicity are required for this purpose. Methods Reconstruct the gene of HDV antigen based on the bias code of Escherichia coli, the recombinant protein expresses by high-density fermentation with fed-batch feeding strategy, and purify by immobilized metal chromatography. The sensitivity and specificity of this antigen detect by ELISA method. Results The expression of HDV antigen can reach 20% of the total cell mass in the soluble form. The recombinant HDV antigen can be conveniently purified（98%） by immobilized metal ion affinity chromatography（IMAC） using the interaction between a His-tag and nickel ions. Production of recombinant HDV antigen can reach 0.5 g/L under conditions of high-density cell fermentation. Applied to the diagnostic ELISA method, the recombinant HDV antigen shows excellent sensitivity（97% for IgM and 100% for IgG） and specificity（100% for IgG and IgM） for the detection of anti-HDV antibodies. Conclusion Expression and purification the recombinant HDV antigen as a candidate protein for application in a diagnostic ELISA for HDV infection. Large-scale production of the protein can be achieved using the high-density fermentation strategy.
基金Supported by the National Science and Technology Key Funds (2003DA901A32) and the National Natural Science Foundationof China (No.20476085).
文摘The kinetics of batch and fed-batch cultures of recombinant Escherichia coli producing human-like collagen was investigated. In the batch culture, a kinetic model of a simple growth-association system was concluded without consideration of cell endogeneous metabolism. The cell lag time, the maximum specific growth rate and Yx/s were determined as 1.75h, 0.65h^-1 and 0.51g·g^-1, respectively. In the fed-batch culture, different specific growth rates were set at （0.15, 0.2, 0.25h^-1） by the method of pseudo-exponential feeding, and the expressions for the specific rate of substrate consumption, the growth kinetics and the product formation kinetics of each phase were obtained. The result shows that the concentrations of cell and product can reach 77.5g·L^-1 and 10.2g·L^-1 respectively. The modal predictions are in good agreement with the experimental data.
文摘Several technical parameters were studied during the fermentation of recombinant E.coli for the production of collagen-like biopolymer.The effects of dissolved oxygen as well as glucose concentration on fermentation were observed.The OD 600 value could reach 98 when dissolved oxygen was controlled at 50% and glucose around 1%.The production of human-like collagen with a yield of 29.4% was obtained.
文摘厌氧发酵过程中,物料分层及各层料液黏度、密度等参数是搅拌装置设计的重要依据。该文采用接力试验研究了牛粪中温厌氧发酵过程中各层料液基础物性的变化情况。结果表明:牛粪厌氧发酵过程中,反应器中料液密度自上而下逐渐变大,且各层密度随总固体含量增加而升高,反应过程中各层密度均在发酵第4天达到最大值,料液总固体质量分数为4%、6%、8%时对应的上中下层溶液的密度最大值分别为1.02、1和1.02,1.02、1.03和1.07,1.03、1.03和1.07 g/cm3。受料液分层的影响,反应器中上层和中层料液黏度呈先增大后减小并逐步趋于稳定,下层料液黏度以初始黏度为最大,且不同初始料液总固体含量对黏度变化过程具有显著影响,TS=4%时,上、中层料液黏度分别在第7天、第4天达到最大值11.5和14.7 m Pa·s,下层初始最大黏度为107 m Pa·s;TS=6%、8%时,上、中层料液黏度均在第4天达到最大值,上层料液黏度最大值分别为25.5和63.5 m Pa·s,中层料液黏度最大值分别为15.5和95.5 m Pa·s,下层初始最大黏度分别为135.5和185.5 m Pa·s。随着初始物料总固体含量的增加日产气量也相应增高,产气高峰出现时间相应提前;TS=8%时的累积产气量分别比6%和4%提高了21.4%和8.71%,但产气中甲烷含量增加速率基本相同,并在第10天左右基本趋于稳定。该结果可为厌氧发酵反应器的搅拌装置设计提供参考。
