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Prokaryotic expression and purification method and application of PK34 antibacterial peptide

A purification method and prokaryotic expression technology, applied in the field of prokaryotic expression and purification of PK34 antimicrobial peptides, can solve the problems of long production cycle, high cost, increased treatment cost, etc., and achieve low production cost, short production cycle and high product quality. Effect

Pending Publication Date: 2022-07-29
WEIFANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method has the problems of high cost, long production cycle, low protein yield, and uneven folding. If the polypeptide is used in clinical treatment later, the cost of treatment will be greatly increased

Method used

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  • Prokaryotic expression and purification method and application of PK34 antibacterial peptide
  • Prokaryotic expression and purification method and application of PK34 antibacterial peptide
  • Prokaryotic expression and purification method and application of PK34 antibacterial peptide

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] A prokaryotic expression and purification method of PK34 antimicrobial peptide,

[0048] step one:

[0049] The codon optimization software MaxCodonTM Optimization Program (V13) optimizes the amino acid sequence of the provided PK34 protein, adopts the whole gene synthesis and inserts the PK34 gene into the expression vector pSumo-DT through the restriction enzyme sites StuI and HindIII, and through the enzyme Excision and sequencing confirmed the accuracy of the final expression vector. To facilitate purification, His-SUMO-tag was designed at the N-terminus of the recombinant polypeptide.

[0050] Step 2:

[0051] (1) Expression vector transformation and induced expression:

[0052]The constructed plasmid containing the PK34 gene was transformed into BL21 (DE3) competent cells, and then evenly spread on LB plates (containing 50 μg / mL kanamycin sulfate), and then placed in an incubator at 37°C overnight. Pick a single clone from the transformed plate, inoculate it i...

Embodiment 2

[0070] PK34 protein detection:

[0071] Step 1: PK34 protein stability test (freeze-thaw experiment):

[0072] Take a piece of PK34 protein frozen at -80°C, put it in an ice-water bath for 5-10min to slowly thaw, and then place it in a 4°C refrigerator for 0.5h after thawing. There is no abnormal phenomenon, indicating that the protein freeze-thaw experiment is normal.

[0073] Step 2: Determination of PK34 protein concentration:

[0074] The protein concentration was measured using a Bradford protein concentration assay kit, and the protein concentration was 0.4 mg / mL.

[0075] Step 3: Determination of PK34 protein purity:

[0076] Determination of protein purity using SDS-PAGE method, BSA as the standard, purity > 85%, evaluation derived from R250 staining SDS-PAGE, such as Figure 5 Indicated, where Lane M: SDS-PAGE Marker; Lane 1: PK34 protein (1.50 μg); Lane 2: BSA (2.00 μg).

[0077] Step 4:

[0078] LC-MSMS protein identification:

[0079] (1) Enzymatic hydrolysis...

Embodiment 3

[0099] PK34 antibacterial effect verification:

[0100] The antibacterial effect detection method was a microdilution method based on resazurin redox colorimetry.

[0101] First, take a clean internal spiral glass tube, mix H37Rv in log phase with 7H9 medium containing 10% OADC, and adjust to 1 McFarland concentration (about ~10 7CFU / mL), note that the bacterial suspension should not contain bacterial clumps. Then mix the bacterial suspension with 7H9 medium containing 10% OADC at a ratio of 1:25, and dilute to about 5x10 5 CFU / mL bacterial suspension.

[0102] Set control group: ①Blank control group was 200uL 7H9 medium containing 10% OADC; ②positive control group was 100uL bacterial suspension+100uL 7H9 medium containing 10%OADC; ③negative control group was 100uL bacterial suspension+100uL 7H9 medium containing 10% OADC, and then 1uL of rifampicin stock solution (8mg / mL) was added to make the final concentration of rifampicin to be 40 ug / mL; ④ Resazurin blank control grou...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a prokaryotic expression and purification method and application of a PK34 antibacterial peptide. The method comprises the following steps: 1, optimizing a PK34 polypeptide amino acid sequence, inserting a PK34 gene into an expression vector through a restriction enzyme cutting site by adopting whole-genome synthesis, and constructing a recombinant expression plasmid; 2, the constructed recombinant expression plasmid containing the protein of the PK34 gene coding sequence is transformed into a prokaryotic cell, and PK34 polypeptide is expressed through induction; 3, splitting the expressed prokaryotic cells, and detecting expression positions in a supernatant and a precipitate; and 4, purifying the PK34 protein. The simple and convenient method for rapidly preparing the PK34 polypeptide in a large scale is achieved, the target polypeptide which is high in purity and still has antibacterial activity without treatment is obtained through the steps of large-scale expression of the PK34 polypeptide in prokaryotic cells and simple and convenient purification, the production period is short, the production cost is low, the protein yield is high, the product quality is high, and the method is suitable for industrial production. And the method can be used for preparing the anti-PK34 monoclonal antibody.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a prokaryotic expression and purification method and application of a PK34 antimicrobial peptide. Background technique [0002] Although tuberculosis can be prevented and cured by chemical drugs, due to the shortcomings of chemotherapy drugs with strong toxic and side effects, and the long time of anti-TB treatment and the combination of multiple chemotherapy drugs, MTB is prone to multi-drug resistance and extensive drug resistance. Therefore, the development of new anti-MTB infection and lesion repair methods is of great value. [0003] As a biological antibacterial drug, antimicrobial peptides have a series of advantages themselves: extensive existence, broad antibacterial spectrum, rapid sterilization, no accumulation of toxic effects, etc. Because antimicrobial peptides have different mechanisms of action from traditional antimicrobials, they are an important candidate for comb...

Claims

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Application Information

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IPC IPC(8): C07K14/00C07K1/22C12N15/70C12N15/66C12P21/06A61K38/16A61P31/06
CPCC07K14/00C12N15/70C12N15/66C12P21/06A61P31/06C12N2800/22C12N2800/101A61K38/00
Inventor 李倩王佳齐袁廷勋孙鼎淳高伟
Owner WEIFANG MEDICAL UNIV
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