Method for detecting specific recombinant human insulin escherichia coli residual host protein

A technology for recombinant human insulin and human insulin, applied in biological testing, material inspection products, etc., can solve the problems of inapplicable residual host protein detection, poor sensitivity, and measurement results that cannot truly reflect residual host protein

Active Publication Date: 2013-12-11
TONGHUA DONGBAO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ELISA kits are currently used for the detection of residual host proteins of E. coli products in RHI products. The antigen used in this kit to prepare antibodies is common E. coli, so when detecting the residual host proteins of RHI products with specific processes, specificity Low, poor sensitivity, the test results cannot truly reflect the content of residual host proteins in the product, even if the test results are qualified, it cannot fully explain its safety
In addition, up to now, common assay methods (such as high performance liquid chromatography, etc.) are not suitable for the detection of residual host proteins in RHI with process specificity

Method used

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  • Method for detecting specific recombinant human insulin escherichia coli residual host protein
  • Method for detecting specific recombinant human insulin escherichia coli residual host protein
  • Method for detecting specific recombinant human insulin escherichia coli residual host protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] 1.1 Preparation of Escherichia coli containing the pZRHi-1 plasmid integrated with the human insulin gene:

[0095] 1.1.1 The human proinsulin gene sequence was synthesized by the solid-phase phosphoramidite method, and the human proinsulin gene sequence contained a 5'Cla I and a 3'Hind III site;

[0096] 1.1.2 Add Cla I and Hind III restriction endonuclease 0.005M to 0.01M pZRHi-1 plasmid and 0.01M human proinsulin gene containing a 5'Cla I and a 3'Hind III site respectively, at 37 ℃ for 1 hour, then keep at 60℃ for 10-15 minutes to terminate the reaction; mix the two reaction solutions, add DNA ligase 0.005M, react at 16℃ for 11 hours, and obtain a recombinant plasmid capable of expressing the human insulin gene;

[0097] 1.1.3 Add calcium chloride to the culture medium of Escherichia coli at a ratio of 100:1 (V / W), and then add the recombinant plasmid obtained in the above steps at a ratio of 1:1 (V / V), and place on ice at 4°C Bath (also called a water bath) for 30 ...

Embodiment 2

[0136] 1.1 Preparation of Escherichia coli containing the pZRHi-1 plasmid integrated with the human insulin gene:

[0137] 1.1.1 The human proinsulin gene sequence was synthesized by the solid-phase phosphoramidite method, and the human proinsulin gene sequence contained a 5'Cla I and a 3'Hind III site;

[0138] 1.1.2 Add 0.05M of Cla I and Hind III restriction endonucleases to 0.01M pZRHi-1 plasmid and 0.01M human proinsulin gene containing a 5'Cla I and a 3'Hind III site, respectively. Act at 35°C for 1 hour, then keep at 65°C for 10 minutes to terminate the reaction; mix the two reaction solutions, add DNA ligase 0.005M, and react at 14°C for 10 hours to obtain a recombinant plasmid expressing the human insulin gene;

[0139] Calcium chloride is added to the culture solution of Escherichia coli, and the recombinant plasmid obtained in the above steps is added,

[0140] 1.1.3 Add magnesium sulfate to the culture medium of Escherichia coli at a ratio of 100:1 (V / W), then add...

Embodiment 3

[0179] 1.1 Preparation of Escherichia coli containing the pZRHi-1 plasmid integrated with the human insulin gene:

[0180] 1.1.1 The human proinsulin gene sequence was synthesized by the solid-phase phosphoramidite method, and the human proinsulin gene sequence contained a 5'Cla I and a 3'Hind III site;

[0181] 1.1.2 Add 0.005M Cla I and Hind III restriction enzymes to the 0.01M pZRHi-1 plasmid and the 0.01M human proinsulin gene containing a 5'Cla I and a 3'Hind III site, respectively. Act at 35°C for 1 hour, then keep at 60°C for 12 minutes to terminate the reaction; mix the two reaction solutions, add DNA ligase 0.005M, and react at 18°C ​​for 12 hours to obtain a recombinant plasmid expressing the human insulin gene;

[0182] 1.1.3 Add calcium chloride to the Escherichia coli culture solution at a ratio of 100:1 (V / W), then add the recombinant plasmid obtained in the above steps at a ratio of 1:1 (V / V), and place on ice at 2°C Bath for 25 minutes, then place in a 45°C wa...

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Abstract

The invention relates to a method for detecting specific recombinant human insulin escherichia coli residual host protein, which comprises the steps of taking total host protein prepared by ultrafiltration of escherichia coli fermentation liquid integrating pZRHi-1 plasmid of a human insulin gene as an antigen, stimulating a rabbit to generate an antibody, removing a human insulin antibody by affinity chromatography fixed with human insulin to prepare a specific antibody, labeling the specific antibody with horse radish peroxidase, and then detecting the residual host protein for a sample to be detected by a double antibody sandwich method. The method is high in sensitivity and specificity, and good in repeatability, and can effectively control RHI (recombinant human insulin) product quality.

Description

technical field [0001] The invention relates to a detection method for recombinant human insulin E. coli residual host protein, in particular for detecting the content of E. coli residual host protein of process-specific recombinant human insulin produced by Tonghua Dongbao Pharmaceutical Co., Ltd. Background technique [0002] Recombinant human insulin (hereinafter referred to as RHI) has been widely used in the clinical treatment of type II diabetes worldwide since it was approved by the US FDA in 1982. RHI is usually prepared by Escherichia coli engineering bacteria. Just like other recombinant proteins, it is almost inevitable that the host protein will remain in the product, causing contamination to the product. When the cumulative concentration of these residual host proteins in the blood reaches 100ppm, it is likely to stimulate the human immune system to cause an immune response, thereby affecting the efficacy of the drug. [0003] At present, RHI products for clini...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
Inventor 冷春生
Owner TONGHUA DONGBAO PHARMA
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