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
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.
