Antibacterial protein and preparation method and application thereof

An antibacterial protein and reaction technology, applied in the fields of biochemistry and molecular biology, can solve the problems of antibacterial protein’s killing effect on bacteria, low content of natural antibacterial protein, and inability to meet application needs, so as to prevent and treat infectious diseases, reduce Production costs, effects of overcoming adverse consequences

Inactive Publication Date: 2013-09-18
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The content of natural antibacterial protein is very low, and the extraction steps are complicated; the cost of preparing protein by chemical synthesis is very high, which is far from meeting the application needs; antibacterial protein has a killing effect on bacteria, so it is not suitable for direct expression in prokaryotic systems

Method used

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  • Antibacterial protein and preparation method and application thereof
  • Antibacterial protein and preparation method and application thereof
  • Antibacterial protein and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Embodiment 1: Synthesis contains the DNA product of antibacterial protein gene

[0027] According to the amino acid sequence of the antibacterial protein, the gene sequence was designed (as shown in sequence 2 in the sequence listing), and four pairs of primers were synthesized with a DNA synthesizer: primer P1 shown in sequence 3 in the sequence listing and primer R1 shown in sequence 4 in the sequence listing , primer P2 shown in sequence 5 in the sequence listing and primer R2 shown in sequence 6 in the sequence listing, primer P3 shown in sequence 7 in the sequence listing and primer R3 shown in sequence 8 in the sequence listing, sequence 9 in the sequence listing The primer P4 shown and the primer R4 shown in sequence 10 in the sequence listing. Primer P4 introduces an EcoRI restriction site (GAATTC) and a methionine codon (ATG), and primer R4 introduces a stop codon and an EcoRI restriction site. Methionine was introduced to remove the increased N-terminal fusio...

Embodiment 2

[0032] Example 2: Construction of pPIC9K recombinant plasmid containing antibacterial protein gene. The specific process is:

[0033] (1) Digest the DNA product in step (4) of Example 1 with EcoRI.

[0034] (2) The pPIC9K plasmid was digested with EcoRI and dephosphorylated with CIAP alkaline phosphatase.

[0035] (3) Electrophoresis and recovery of the digested DNA product and pPIC9K, ligation with T4 DNA ligase at 16°C for 4 hours, transformation of the ligation product into Escherichia coli TOP10 strain, selection of single colony on LB medium containing ampicillin resistance, and expansion culture Plasmids were then extracted.

[0036] (4) The PCR method was used to screen the pPIC9K recombinant plasmid inserted into the antimicrobial protein gene. The PCR reaction conditions are: 1 μL of the plasmid to be detected, 1 μL each of primer α-Factor shown in sequence 12 in the sequence listing and primer R4 (10 μmol / L) shown in sequence 10 in the sequence listing, 10 μL of 2...

Embodiment 3

[0039] Example 3: Synthesis of the DNA product of the antimicrobial protein gene with histidine tag. The specific process is:

[0040] The steps are the same as in Example 1, except that primer R5 shown in sequence 13 in the sequence listing is used in step (4) to replace primer R4 shown in sequence 10 for PCR amplification to obtain the antimicrobial protein gene with histidine tag DNA product (see sequence 14 in the sequence listing), the process is as follows figure 1 shown. Primer R5 introduced a codon encoding six histidines (CATCACCATCACCATCAC), a stop codon (TAA) and an EcoRI restriction site (GAATTC).

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Abstract

The invention provides an antibacterial protein and a gene for coding the antibacterial protein, and also provides a preparation method and application of the antibacterial protein. The antibacterial protein provided by the invention has antibacterial activity to Gram-positive bacteria, does not have hemolytic activity to human red blood cells, and has good safety. In the invention, by selecting the optimized codon suitable for yeast expression, the constructed antibacterial protein gene can be efficiently expressed in pichia pastoris, the industrialization can be realized, and the production cost can be lowered. The antibacterial protein with a histidine label can be quickly purified through a nickel column, thereby simplifying the preparation scheme. The antibacterial protein provided by the invention can be used for preparing antibacterial drugs and sanitizers. After the antibacterial protein provided by the invention is used for resisting bacteria and sanitizing germs, the negative effects caused by abuse of antibiotics can be overcome, the effects of preventing and treating infectious diseases and sanitizing germs can be achieved, and the production cost can be lowered.

Description

technical field [0001] The invention belongs to the field of biochemistry and molecular biology, relates to antibacterial protein, in particular to the amino acid sequence of antibacterial protein and its preparation method and application. Background technique [0002] Antibiotics are the main weapon for humans to resist bacterial infections and diseases, and play an important role in the history of medical development. However, with the long-term use and abuse of antibiotics, the resistance of many bacteria has increased, such as clinically methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, etc. The recent emergence of "super bacteria" can tolerate almost all antibiotics, and there have been many fatal cases, causing worldwide panic. It is imminent to find drugs that are free from bacterial resistance and can replace antibiotics. [0003] Antimicrobial proteins are small proteins produced by organisms induced by immunity, and have multiple functi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00C12R1/84
Inventor 陆婕
Owner HUAZHONG UNIV OF SCI & TECH
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