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A visual antimicrobial peptide fusion protein and its preparation method and application

An antibacterial peptide and recombinant protein technology, applied in the field of group engineering, can solve the problems of consuming a lot of manpower and material resources, protease attack, affecting the expression efficiency, etc., to achieve good application prospects, increase expression, and reduce the effect of possibility

Active Publication Date: 2021-07-20
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since antimicrobial peptides are basic polypeptides with small molecules containing multiple basic amino acids, they are extremely vulnerable to protease attacks
The products expressed at the same time are often toxic to the host cells, which will directly inhibit the growth of the host bacteria and affect the expression efficiency
Therefore, the exogenous expression of antimicrobial peptides is more difficult than other peptide drugs
In addition, when screening high-efficiency engineering bacteria for antimicrobial peptides, antimicrobial peptides are usually used to have special antibacterial activity. After screening by antibacterial experiments, thousands of transformants must be identified each time. Carry out preliminary screening, use antibacterial screening of engineering bacteria with relatively high antibacterial activity, and then carry out rescreening, which will consume a lot of manpower and material resources
Therefore, how to protect antimicrobial peptides from protease attacks, reduce their toxicity to host cells, and how to screen yeast engineering bacteria that express antimicrobial peptides efficiently have become bottlenecks in the development of antimicrobial peptides.

Method used

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  • A visual antimicrobial peptide fusion protein and its preparation method and application
  • A visual antimicrobial peptide fusion protein and its preparation method and application
  • A visual antimicrobial peptide fusion protein and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Embodiment 1, red fluorescent protein mApple gene acquisition and sequence analysis

[0077] According to the known sequence of the gene mApple, using the codon preference of Pichia pastoris, the sequence was optimized and transformed without changing its amino acid sequence, and a his purification tag was added at the 5' end, and the sequence was sent to the company for synthesis. The red fluorescent protein mApple contains 236 amino acids, plus 6 histidines, a total of 242 amino acids, and the theoretical molecular weight of the mature protein is 28kDa. The amino acid sequence of the red fluorescent recombinant protein his-mApple with his purification tag added is shown in the sequence table Shown in SEQ ID NO.1;

[0078] The nucleotide sequence of the coding gene of the red fluorescent recombinant protein his-mApple that is modified by optimizing the codon bias of Pichia pastoris and added with the his purification tag is shown in SEQ ID NO.2 in the sequence listing....

Embodiment 2

[0085] Example 2, the gene of honeybee antimicrobial peptide AP precursor and the synthesis of DP dipeptide acid cleavage site

[0086] According to the known sequence of honeybee antimicrobial peptide AP gene, according to the codon preference of Pichia pastoris, the nucleotide sequence was optimized without changing its amino acid sequence, and the gene coding for DP dipeptide was added at its 5' end Sequence: GACCCA, send the company to synthesize the nucleotide sequence. The honeybee antimicrobial peptide AP has a total of 18 amino acids, and the theoretical molecular weight of the mature protein is 2.2kDa. The amino acid sequence of the antimicrobial peptide AP is shown in SEQ ID NO.3;

[0087] The nucleotide sequence encoding the honeybee antimicrobial peptide AP modified by optimizing the codon preference of Pichia pastoris is shown in SEQ ID NO.4 in the sequence listing.

[0088] The nucleotide sequence encoding the honeybee antimicrobial peptide AP of the gene coding...

Embodiment 3

[0093] Embodiment 3, the acquisition of the fusion gene his-mApple-DP-AP

[0094] The PCR product A obtained in Example 1 and the PCR product obtained in Example 2 were used as a common template, and mApple-F1 and AP-R were used as primers for PCR amplification.

[0095]The PCR product B obtained in Example 1 and the PCR product obtained in Example 2 were used as a common template, and mApple-F2 and AP-R were used as primers for PCR amplification.

[0096] The PCR reaction parameters were: 95°C for 5 min; 32 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 90 sec; 72°C for 10 min. PCR Product Purification Kit to purify PCR products.

[0097] The sequencing results of the above PCR amplification products show that the two nucleic acid fragments obtained by the above PCR amplification respectively include XhoI, Not I double restriction sites and EcoR I, Not I double restriction sites, respectively named his-mApple-DP -AP-1 and his-mApple-DP-AP-2, the middle of the two ...

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Abstract

The invention mainly provides a method for highly expressing antimicrobial peptides in yeast. The method provided by the present invention comprises the fusion expression of an antimicrobial peptide and a red fluorescent protein, and in the recombinant yeast, the level of antimicrobial peptide production in the strain can be judged by the color depth visible to the naked eye, so as to quickly screen out highly expressed antimicrobial peptides and Engineering strains with strong antibacterial activity. The invention also provides a recombinant fusion protein with antibacterial activity and engineering recombinant bacteria, which can be directly applied in industry or feed industry.

Description

technical field [0001] The invention belongs to the field of group engineering. Specifically, the invention relates to a visualized antimicrobial peptide fusion protein, its preparation method and its application. Background technique [0002] Antibacterial peptides are cationic (containing too much lysine and arginine) amphiphilic small molecular polypeptides generally composed of 12 to 50 amino acid residues. Widely present in animals, plants and microorganisms, it is an important part of the host immune defense system. It has a wide antibacterial spectrum, low immunogenicity, and is not easy to produce drug resistance and has the effect of inhibiting the growth of cancer cells. This makes antibacterial The development and utilization of peptides has become a research hotspot in recent years. However, the development and utilization of antimicrobial peptides is not easy. The natural resources of antimicrobial peptides are limited, and the extraction is very complicated; c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/81
CPCC07K5/06C07K14/43572C07K14/43595C07K2319/21C12N15/815
Inventor 姚斌黄火清华婷罗会颖王苑柏映国孟昆涂涛王亚茹苏小运
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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