A method for non-methanol-induced production of antimicrobial peptides

An antibacterial peptide and methanol technology, applied in the fields of bioengineering and biopharmaceuticals, can solve the problems of inapplicability of antibacterial peptide products, unsuitable food or additive production, poor expression effect of antibacterial peptides, etc., and achieve the effect of broad application value and market prospect.

Active Publication Date: 2020-08-14
深圳市微宇生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the current Pichia pastoris expression system faces many problems in the fermentation and amplification process: methanol is used as a carbon source when inducing the expression of exogenous recombinant proteins. Methanol is toxic, not suitable for the production of food or additives, and is flammable and explosive. It is easy to be volatile, and the workshop needs to be anti-riot design in industrial production, which leads to increased costs; methanol fermentation consumes a lot of oxygen, and it is generally difficult to meet the demand for oxygen by increasing the air volume and speed of the air, so it is necessary to pass a lot of pure oxygen. Industrialized production brings inconvenience. In addition, the more methanol consumed, the greater the heat produced, and the higher the cooling capacity of the required equipment; the efficient transcription of the Pichia pastoris AOX1 promoter cannot do without methanol, which suffers from other carbon sources. carbon source repression
[0007] These problems lead to poor expression of antimicrobial peptides in Pichia pastoris. More importantly, methanol is retained in the antimicrobial peptide products induced by methanol. The toxicity of methanol makes this antimicrobial peptide product unable to be used in the fields of food, feed and medicine.

Method used

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  • A method for non-methanol-induced production of antimicrobial peptides
  • A method for non-methanol-induced production of antimicrobial peptides
  • A method for non-methanol-induced production of antimicrobial peptides

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0054] Amplification of Example 1 FLD1 Promoter and Construction of PisL9K22WK Gene Expression Cassette

[0055] According to the AA sequence of the antimicrobial peptide PisL9K22WK, the gene encoding the antimicrobial peptide PisL9K22WK was designed, and the DNA sequence after codon optimization was as follows:

[0056] ATG TTC TTC TTC CAC ATC ATC AAG GGT AAG TTC CAC GCT GGT AGA ATG ATCCAC GGT TTG GTC TGG AAG TAA (SEQ ID NO. 3)

[0057] PCR amplification of FLD1 promoter and PisL9K22WK gene expression cassette

[0058] FLD5BZ (Seq ID NO.4): 5'-GCGAGATCTGCATGCAGGAATCTCTGG-3';

[0059] FLD3HZ (Seq ID NO.5): 5′-GCGGAATTCTGTGAATATCAAGAATTGTATGAACAAGC-3′;

[0060] pis3R (Seq ID NO.6):

[0061] 5'-CCAGCGTGGAATTTGCCTTTGATTATATGAAAGAAGAACATAGGCCTTGTGAATATCAA-3';

[0062] pis3R-pis (Seq ID NO.7): 5'-aatagtGGTACCtcattaTTTCCAGACCAAACCATGAATCATCCTACCAGCGTGGAATTTGCCT-3';

[0063] Using the yeast genome as a template, the reaction system is as follows:

[0064] The PCR reaction syste...

Embodiment 2

[0080] Example 2 Construction of Pichia pastoris intracellular expression vector of antimicrobial peptide PisL9K22WK

[0081] 2.1 The FLDPis-T obtained in Example 1 was double digested with BglII and KpnI endonucleases. At the same time, the pPinkLC vector (purchased from Invitrogen) was double digested with BglII and KpnI.

[0082] The double enzyme digestion system is as follows:

[0083]

[0084] Digestion conditions: 37°C water bath for 1h.

[0085] The digested products were recovered with a gel extraction kit (purchased from TAKARA), and stored at -20°C for future use. Both FLDPis-T and pPinkLC vectors were digested with Bgl II and Kpn I, and ligated with T4 DNA ligase.

[0086] The connection system is as follows:

[0087]

[0088] Ligation conditions: 22°C, 1h; 16°C, 1h; 4°C overnight.

[0089] 2.2 Transform the obtained expression vector into Escherichia coli DH5α: Take out 100 μl of Escherichia coli DH5α competent cells and freeze them at -80°C, place them ...

Embodiment 3

[0101] The construction of embodiment 3PisL9K22WK engineering bacteria

[0102] 3.1 Linearization of expression vector FPLC

[0103] The expression vector FPLC obtained in Example 2 was linearized with SpeI endonuclease and used for PichiaPink yeast transformation.

[0104] The linearization system is as follows:

[0105]

[0106] Reaction conditions: 37°C, 14h.

[0107] Figure 4 It is the electrophoresis result of the expression vector FPLC linearized by SpeI digestion in Example 3: among them, M: 1kb DNAmarker, the sample volume is 5 μl; 1: the expression vector FPLC that has not been linearized, the sample volume is 10 μl; 2: linear The expression vector FPLC of the culture, the sample volume is 10 μ l; Electrophoresis result ( Figure 4 ) shows that the expression vector FPLC of the antimicrobial peptide PisL9K22WK is completely linearized.

[0108] 3.2 Preparation of PichiaPink Yeast Competent Cells

[0109] Pick a single colony of PichiaPink yeast (purchased fr...

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Abstract

The invention discloses a method for producing antibacterial peptide through non-methanol induction. The invention relates to the technical field of the bioengineering and bio-pharmaceuticals, the bad expression effect of the antibacterial peptide in the pichia pastoris is solved, the most important is the methanol resides in the antibacterial peptide product expressed by use of the methanol induction, the toxicity of the methanol enables the antibacterial peptide product to be not applied in the fields of the food, the fodder and the medicines. The technical scheme provided by the invention is to add the inductor choline chloride into an inoculated culture medium so that the final concentration of the choline chloride in the culture medium is 1%-5%w / v. The method disclosed by the invention has the advantages that the PisL9K22WK expression level is high, the production cost is low, the product is safe and free from poisonous residue; the engineering saccharomycetes and the produced pisL9K22WK product can be applied to the antibacterial medicines or the animal or aquiculture fodder additives.

Description

technical field [0001] The invention relates to the technical fields of bioengineering and biopharmaceuticals, and more particularly relates to a method for inducing the high-efficiency expression of the improved grouper antimicrobial peptide pisL9K22WK in Pichia pink by using a non-toxic inducer. Background technique [0002] Antimicrobial peptides (AMP) are positively charged, cation-rich, and amphiphilic small molecule polypeptides in organisms. The main characteristics are as follows: high antibacterial activity, wide antibacterial spectrum, and many types. It is not easy to produce drug resistance to bacteria. The post-metabolism of the body is fast, it is not easy to produce harmful residues in the body, and it has killing or inhibiting effects on fungi, viruses, protozoa and even tumors. Due to its outstanding biological characteristics, it has the most potential as a new type of safe and harmless antibiotic substitute, and has attracted the attention of the scientifi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/00C12N15/81C12N1/19C12N15/12C12R1/84
CPCC07K14/461C12N15/815C12N2800/102
Inventor 胡章立贾彬雷宇邓利
Owner 深圳市微宇生物科技有限公司
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