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Radioresistant peptide mutant protein and its preparation and purification method

A technology for mutant proteins and purification methods, which is applied in the fields of peptide preparation methods, chemical instruments and methods, and botanical equipment and methods, can solve the problems of insufficient consideration of the structural integrity and protein folding properties of flagellin proteins, and achieve easy Preservation, reduce degradation rate, improve the effect of purity

Active Publication Date: 2022-03-15
ACADEMY OF MILITARY MEDICAL SCI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The radioantipeptide protein has an artificially designed linking sequence inserted between its ND1 and CD1 domains and retains part of the amino acid sequence of the CD2 domain. This molecular structure does not fully consider the integrity of the flagellin protein structure and protein folding characteristic

Method used

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  • Radioresistant peptide mutant protein and its preparation and purification method
  • Radioresistant peptide mutant protein and its preparation and purification method
  • Radioresistant peptide mutant protein and its preparation and purification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Preparation and purification of radioresistant peptide muteins

[0030] 1) Construction of radioresistant peptide mutein expression vector

[0031] Synthesize the cDNA of the radioresistant peptide mutein according to the nucleotide sequence shown in SEQ ID NO: 2 by base synthesis method, and connect it to the pBV220 temperature-inducible expression vector after restriction enzyme digestion to construct the pBV220-FKT recombinant expression vector , see the results of vector digestion and identification figure 1 , and then use this vector to transform Escherichia coli JM109, screen positive clones on ampicillin-resistant LB medium, and establish a seed bank of engineering bacteria.

[0032] 2) Fermentation and electrophoresis identification of radioresistant peptide mutant protein

[0033] The JM109 engineered bacteria containing the recombinant bacterial flagellin-derived polypeptide radioresistant peptide mutant carrier were shaken and cultured overnight a...

Embodiment 2

[0048] Example 2 Preparation and Purification of Unmutated Radioresistant Peptide Protein CBLB502

[0049] The preparation method of the radioresistant peptide protein CBLB502 is similar to Example 1, the difference is that when constructing the expression vector, the cDNA of the radioresistant peptide protein CBLB502 is synthesized in vitro by base synthesis method, and connected to the temperature-inducible expression vector In pBV-220; then the JM109 engineering bacteria containing the recombinant bacterial flagellin-derived polypeptide radioantipeptide protein CBLB502 carrier were fermented, expressed and purified according to the conditions of step 2)-step 6), and electrophoresis analysis was carried out, and the results showed that the present invention The method is also applicable to the preparation and purification of unmutated radioresistant peptide protein ( Figure 9 ).

Embodiment 3

[0050] Example 3 Biological Activity Assay of Radioresistant Peptide Mutant Protein

[0051] Select the positive clone with the best response to NF-kB stimulating activity to inoculate a 96-well plate, the inoculation medium is DMEM containing only 10% fetal bovine serum without hygromycin B, and the number of cells per well is 1×10 4 , a total of 8 groups, each with 3 replicate wells. After 24 hours, the radioresistant peptide mutant protein was stimulated, and the administration concentration was 10 -12 ~10 -5 g / ml, after 6 hours, detect the value of the luciferase reporter gene ( Figure 10 ).

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Abstract

The invention discloses a method for preparing and purifying a radioresistant peptide or a mutant protein thereof. The method can significantly improve the protein purity and is convenient for industrial production. The present invention also provides a radioresistant peptide mutein, which is based on the radioresistant peptide protein CBLB502, and replaces the amino acid sequence between the ND1 and CD1 domains, and the mutein has SEQ ID NO: The stability of the amino acid sequence shown in 2 is significantly better than that of the CBLB502 protein in the prior art.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a preparation method for the expression, separation and purification of radioresistant peptide protein CBLB502 or its mutein, and to the radioresistant peptide mutein obtained by the above method. Background technique [0002] Toll-like receptors (Toll-like receptors, TLRs) were discovered by Lemaitre et al. in the study of Drosophila in 1996. Current research shows that 11 members of the TLRs family have been found in the human body, mainly expressed in immune cells. The Toll-like receptors (TLRs) family is a class of pattern recognition receptors that play an important role in the body's innate and acquired immune responses. They activate the NF-κB signaling pathway through specific recognition of pathogen-related molecular patterns such as bacteria and viruses. Induce inflammatory response, antiviral response and differentiation and maturation of immune effector cells, and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/255C07K1/20C07K1/18C12N15/31C12N15/70C12N1/21C12R1/19
CPCC07K14/255
Inventor 葛常辉杨刚刚杨晓明张全义张全海詹轶群王磊吕中原王海滨
Owner ACADEMY OF MILITARY MEDICAL SCI
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