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A method for promoting in vitro refolding and improving immunogenicity of low-molecular-weight proteins

An immunogenic and external renaturation technology, which is applied in the preparation method of peptides, chemical instruments and methods, immunoglobulin, etc., can solve the problem of incorrect protein structure, PEG and its derivatives cannot be renatured with antigen molecules, antibody Problems such as excessive difference in nature

Active Publication Date: 2022-05-10
北京春雷杰创生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Immunization with low-molecular-weight protein antigens often faces two problems: 1. The immunogenicity of low-molecular-weight proteins is low, and it is difficult to obtain high-titer antibodies; 2. The structure of the protein is incorrect, and the properties of the antibodies produced are too different from those produced by natural proteins.
Both solutions have their own advantages and insurmountable disadvantages, so they cannot be used alone
[0016] Therefore, we need to develop an antigen refolding scheme in vitro, which can not only solve the problem that PEG and its derivatives cannot be used for refolding antigen molecules, but also prepare antigens with high immunogenicity, which can be used to prepare high-efficiency Valence, highly sensitive antibody

Method used

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  • A method for promoting in vitro refolding and improving immunogenicity of low-molecular-weight proteins
  • A method for promoting in vitro refolding and improving immunogenicity of low-molecular-weight proteins
  • A method for promoting in vitro refolding and improving immunogenicity of low-molecular-weight proteins

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Experimental program
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Effect test

Embodiment 1

[0048] Using maltose binding protein (MBP) as carrier protein, construct the expression plasmid of renaturation tool molecule according to the requirements of 1.1. The truncation of von Willebrand Factor (vWF) with a molecular weight of 15Kd was used to construct an expression plasmid according to the requirements of 1.2. The ELP sequence used was -VPGAG-VPGAG-. The antigen was obtained for immunizing New Zealand big ear white to prepare polyclonal antibodies. Elisa detection of the immunization process showed that with the increase of the number of immunizations, the antiserum titer of the antigen experimental group containing ELP label was significantly higher than that of the antigen control group without ELP label . Compared with the control group without ELP label, the antiserum titer increased by about 4 times to 1:3000 (e.g. Image 6 shown). The antigen fused with ELP tag achieved better immune effect than conventional renatured antigen.

Embodiment 2

[0050] Using glutathione-S-transferase (GST) as the carrier protein, construct the expression plasmid of the renaturation tool molecule according to the requirements of 1.1. The truncation of Heparin-Binding Protein (HBP) with a molecular weight of 17Kd was used to construct an expression plasmid according to the requirements of 1.2. The ELP sequence used was -VPGSG-VPGSG-VPGSG-. Antigens were obtained and used to immunize BALB / C mice to prepare monoclonal antibodies. Elisa detection of the immunization process showed that with the increase of the number of immunizations, the antiserum titer of the antigen experimental group containing ELP tags was significantly higher than that of the antigens without ELP tags test group. Compared with the control group without ELP label, the antiserum titer increased by 5 times to 1:10000 (e.g. Figure 7 shown). The antigen fused with ELP tag achieved better immune effect than conventional renatured antigen.

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Abstract

This application discloses a method for promoting in vitro renaturation of low-molecular-weight proteins and improving immunogenicity, which mainly includes the following steps: constructing carrier proteins and target proteins containing ELP tags; using modified carrier proteins to prepare renaturation tool molecules; renaturation tool The molecule and the denatured target protein form a renaturation complex through the ELP tag at a temperature higher than the phase transition temperature; after the renaturation complex is completed, the phase transition reaction is induced by lowering the temperature below the phase transition temperature to depolymerize the polymerized ELP tag and release The fully refolded target protein is produced, and the refolded tool molecules are recycled and reused; the fully refolded antigen containing the ELP tag is used to immunize animals, because the temperature of the body is higher than the phase transition temperature, the antigen induces self-aggregation in the body, thereby increasing the antigenic epitope, Improve antigen stability and greatly improve the immune efficiency of low molecular weight proteins. The invention solves the problems that the immunogenicity of the protein after PEG modification decreases so that it cannot be used for immunization and the low molecular weight protein has low immunogenicity and is easily degraded in vivo.

Description

technical field [0001] The present application relates to the field of in vitro renaturation of proteins, and in particular to a method for improving the in vitro renaturation efficiency and immunogenicity of low molecular weight proteins. Background technique [0002] The preparation of antibodies by immunizing experimental animals is a mature biotechnology, and the specific antibodies produced are widely used as tools in life science research, clinical diagnosis, inspection and quarantine and other fields. There are also different requirements for the performance of antibodies according to different application requirements. [0003] In clinical testing, immune response is usually used to detect the content of a target substance in body fluids to diagnose the disease and determine the effect of treatment. Due to the extremely low content of target substances in some cases (such as urine), the relevant antibodies are required to have extremely high titer and sensitivity, a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/113C07K16/00
CPCC07K1/1136C07K16/00
Inventor 郑春杨马鑫申奥
Owner 北京春雷杰创生物科技有限公司