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Polypeptide, HLA-DR protein and preparation method and application of HLA-DR protein

An HLA-DR, protein technology, applied in the field of polypeptide, HLA-DR protein and its preparation, can solve the problems of low yield, low yield, uncontrollable and so on

Inactive Publication Date: 2020-10-23
JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This type of method has the following disadvantages: (1) The content of HLA-DR molecules on the surface of the cell membrane is very small, and even cannot be detected by Western hybridization, so a large number of cultured cells becomes a limiting point in the purification process
(2) During the purification process, complex operations and non-specific adsorption of immunoaffinity columns lead to the incorporation of a small amount of impurity proteins, resulting in insufficient purity; (3) HLA-DR molecules purified from cell lines, most of the antigen-binding sites Both have antigenic peptide binding, which differs from soluble HLA-DR
This type of method has the following disadvantages: (1) The method is cumbersome; (2) DRα and DRβ are assembled in vitro, the yield is low, and the cost is high; (3) The obtained product is a dimer, which is different from the natural soluble HLA-DR
This type of method has the following disadvantages: (1) prokaryotic expression, lack of complex N-glycosylation O-glycosylation and other post-translational modifications, which is very different from the natural protein conformation; (2) in vitro recombination, method Complicated and uncontrollable, low yield, poor operability of the method
[0011] Due to the many disadvantages of the existing extraction and preparation methods of HLA-DR molecules, such as: complex operation, less sources of raw materials, low yield, high cost, and the potential risk of carrying exogenous viruses and other pathogenic microorganisms, it is difficult to realize industrialization , so far, no domestic and foreign reports on products related to the quantitative detection of soluble HLA-DR proteins have been found

Method used

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  • Polypeptide, HLA-DR protein and preparation method and application of HLA-DR protein
  • Polypeptide, HLA-DR protein and preparation method and application of HLA-DR protein
  • Polypeptide, HLA-DR protein and preparation method and application of HLA-DR protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1 HLA-DR protein preparation method (taking SEQ ID NO: 13 as an example)

[0088] 1. Carrier construction

[0089] 1.1. Acquisition of α chain amino acid sequence

[0090] The α chain amino acid sequence and gene sequence of human human leukocyte antigen-DR (HLA-DR) were obtained from the NCBI database (UniProtKB-P01903(DRA_HUMAN); Gene ID: 3122, updated on 16-Feb-2020); HLA - The nucleotide sequence of DRα chain is shown in SEQ ID NO:49.

[0091] 1.2. Acquisition of β chain amino acid sequence

[0092] The β chains are mainly DRB1, DRB3, DRB4, and DRB5, among which DRB1 has the most variation. According to the uniprot database, 50 DRB1, 24 DRB3, 19 DRB4, and 22 DRB5 protein sequences were screened; further comparison and screening were performed to obtain sequences with higher homology, and the β-chain amino acid sequence was designed, with a total of 231 amino acids.

[0093] According to the amino acid sequence of the β chain, the nucleotide sequence of t...

Embodiment 2

[0121] Example 2 Principle and detection steps of sHLA-DR quantitative detection kit

[0122] 1. Inspection principle

[0123] The quantitative detection kit uses the double-antibody sandwich method to detect the content of soluble human leukocyte antigen-DR (sHLA-DR) in plasma. The kit uses a pair of monoclonal antibodies against sHLA-DR (commercially available), one of which is labeled with biotin, and the other is labeled with acridinium ester. The specimen / calibration solution / quality control solution reacts with biotin-labeled monoclonal antibody, acridinium ester-labeled monoclonal antibody, and immunomagnetic particles coated with streptavidin to form an immune complex. The amount of the complex formed It is directly proportional to the content of the antigen to be tested. The acridinium ester in the complex releases photons under the excitation of the substrate solution, automatically monitors the relative light intensity RLU emitted within 3 seconds, and has a certa...

Embodiment 3

[0141] Example 3 Effect of Linker Length Changes on Fusion Protein Activity

[0142] In this embodiment, 6 groups of Linkers with different lengths are designed, as shown in Table 3 below:

[0143] table 3:

[0144]

[0145] Table 4:

[0146]

[0147] The luminescence value in Table 4 is the luminescence value of the sample in the quantitative detection system by diluting the target protein obtained by different design schemes in Table 3 into different concentrations (10000ng / mL, 5000ng / mL, etc.); the membrane protein is the control group. The closer the luminescence value of the isoconcentration sample is to the membrane protein, the better the protein activity; the protein activity is evaluated by calculating the average value of the ratio of the luminescence value of the isoconcentration sample to the membrane protein luminescence value.

[0148] From the above experimental data, it can be seen that when the length of the Linker is 8-48 amino acids, the protein acti...

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Abstract

The invention provides a polypeptide, an HLA-DR protein and a preparation method and application of the HLA-DR protein. The polypeptide is the polypeptide of 8-48 amino acid residues formed by interconnection of glycine and serine through peptide keys. The HLA-DR protein structurally consists of an alpha chain, a beta chain and the polypeptide, wherein the C terminal of the alpha chain is connected with the N terminal of the polypeptide; and the C terminal of the polypeptide is connected with the N terminal of the beta chain. The protein is obtained by connectingn the alpha chain and the betachain which form the HLA-DR protein through 8-48 specific polypeptides consisting of glycine and serine which are in interconnection through peptide keys. The HLA-DR protein which is designed can be in expression in the manner of connecting in series in mammalian cells, can simulate expression of sHLA-DR, and adopts the structure similar to that of natural sHLA-DR. Compared with a film protein mHLA-DR, the HLA-DR protein has more stable conformation, can adopt an eukaryotic expression system for solubiliy high efficiency expression, and is hopeful to have a good application in the field of medical biology and biopharmaceutical development.

Description

technical field [0001] The invention belongs to the technical field of biological immunity, and relates to a polypeptide, an HLA-DR protein, a preparation method and application thereof. Background technique [0002] The major histocompatibility complex (MHC) is a gene group related to the body's immune response. This gene group has more than 100 gene sites, and its coding products include MHC class I molecules and MHC class II molecules. Molecules capable of presenting antigens that play a role in the regulation of immune responses and immune cell development. MHC class II molecules are a group of highly polymorphic transmembrane glycoproteins composed of α-chain and β-chain non-covalent chains presented on the surface of antigen-presenting cells (APC) of the immune system, which can present processed foreign antigens to Antigen receptor for helper T cells, leading to activation and differentiation of helper T cells, which play an important role in inducing immune response...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K14/74C12N15/85C12N5/10G01N33/68
CPCC07K7/08C07K14/70539C12N15/85G01N33/68G01N2333/70539C12N2800/107G01N2800/26
Inventor 马灵芝刘阳张宏航孙玉龙王弢
Owner JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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