CMG 2mutant and Fc fusion protein, its encoding gene and its application

A technology that encodes genes and proteins, applied in gene therapy, antibodies, hybrid peptides, etc., can solve the problems of antibody loss of antigen affinity, difficulty of mouse-derived antibodies, and inability of antibodies to function, and achieve the effect of long half-life

Inactive Publication Date: 2012-06-27
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although vaccination is an effective means to prevent and treat infectious diseases caused by bacteria and viruses, it is not enough to deal with sudden infectious diseases only by vaccination. Taking the prevention and treatment of anthrax as an example, immunization with vaccines has the following deficiencies: (1) Anthrax infections generally arise from emergencies and it is difficult to predict when vaccination will be required
(3) Not all people can produce antibodies after vaccination
(4) The attenuated anthrax vaccine still has certain risks in terms of safety and cannot be widely used
(5) Vaccine alone cannot treat anthrax infection and neutralize anthrax toxin
[0009] There are three difficulties in the development of

Method used

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  • CMG 2mutant and Fc fusion protein, its encoding gene and its application
  • CMG 2mutant and Fc fusion protein, its encoding gene and its application
  • CMG 2mutant and Fc fusion protein, its encoding gene and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0046] Example 1, CMG2 mutant gene design and vector construction

[0047] pIRES2-EGFP-CMG2(E117Q)-Fc is obtained by inserting the CMG2-Fc(E117Q) coding gene (sequence 1 in the sequence list) between XhoI and BamHI of pIRES2-EGFP (Clontech, catalog number: 6029-1) Carrier. The specific construction process is as follows:

[0048] The gene vector pIRES2-EGFP-CMG2-Fc containing the 34-225AA extracellular region of CMG2 is used to insert the CMG2-Fc protein (the amino acid sequence of which is shown in the sequence table 4, and the nucleotide sequence of the sequence 3 in the sequence table) into pIRES2-EGFP (Clontech, catalog number: 6029-1) The vector obtained between XhoI and BamHI. The signal peptide of CMG2-Fc protein is the signal peptide sequence of prourokinase. The 117 position Glu(E) is mutated to Gln(Q), and the 158 is Tyr(Y) to Gln(Q). The mutant is The primers are shown in Table 1. The specific test operations are as follows:

[0049] Using pIRES2-EGFP-CMG2-Fc as a tem...

Embodiment 2

[0060] Example 2. Application of CMG2(E117Q)-Fc

[0061] 1. Protein expression and identification

[0062] 1. Transfection of recombinant mutant gene plasmid into CHO-K1 cells and screening of positive clones

[0063] Transfection uses Lipofectamine produced by Invitrogen TM 2000 cationic liposome transfection kit. According to the kit instructions, the CHO-K1 cells transfected with the vector pIRES2-EGFP-CMG2(E117Q)-Fc obtained in Example 1 (Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences, catalog number: GNHa 7) were cultured in 5% CO 2 , 37℃ incubator. Twelve hours after transfection, the culture was replaced with D-MEM / F-12 medium (Gibco, product number 12500-096) containing 10% peptide bovine serum (Hangzhou Sijiqing company, product catalog number: 4033). After 48 hours, D-MEM / F-12 medium containing 750ug / ml G418 and 10% peptide bovine serum (invitrogen, Product catalog number: 12500-096) Continue to culture, change the medium every 2...

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Abstract

The invention discloses a CMG 2mutant and Fc fusion protein, its encoding gene and its application. The provided protein is any one of the protein in the following 1) or 2) 1) protein composed of an amino acid residue sequence of a sequence 2 in a sequence table; and 2 ) protein derived from 1) by that the amino acid residue sequence of the sequence 2 in the sequence table is subjected to one or more amino acid residue replacement andor deletion andor addition and possesses the same function. The experiment proves that the provided protein can prevent cells from attacking of anthrax toxin, can be taken as anthrax vaccine, the protein and antigen enables high affinity combination, are equivalent to a Fc fragment of the antibody, and the Fc fragment provides the biological activity of the antibody.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular to a fusion protein of CMG2 mutant and Fc and its coding gene and application. Background technique [0002] Anthrax (Anthrax) is a type of infectious disease caused by infection with Bacillus anthracis. Bacillus anthracis is a gram-positive bacillus, and its carriers are herbivores such as horses and deer. Human anthrax infections mainly occur among herders who often come into contact with animals and workers engaged in leather work. There are three ways to cause anthrax infection: one is skin infection, which is mainly caused by the entry of anthrax spores from damaged skin. Bacillus anthracis often accumulates in the damaged skin. This type of infection can often recover without causing serious consequences. The second is gastrointestinal infection, which is often caused by eating raw meat contaminated by spores. This type of infection is often fatal and is also the main mode of...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10C12N15/11A61K39/40A61K48/00A61P31/04
Inventor 郗永义胡显文高丽华岳俊杰邵勇
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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