CN107176979B

CN107176979B - Polypeptide with antibacterial and anti-inflammatory activity and application thereof

Info

Publication number: CN107176979B
Application number: CN201710445058.0A
Authority: CN
Inventors: 强黎明; 卢奎; 董雪茹; 吕名秀; 李玥
Original assignee: Henan Institute of Engineering
Current assignee: Guangdong Shangpinhui Biotechnology Co ltd
Priority date: 2017-06-13
Filing date: 2017-06-13
Publication date: 2020-09-22
Anticipated expiration: 2037-06-13
Also published as: CN107176979A

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a polypeptide with antibacterial and anti-inflammatory activities and application thereof. The nucleotide sequence of the polypeptide is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2. The polypeptide with antibacterial and anti-inflammatory activity is obtained by improving the amino acid structure of the polypeptide Lunasin, can be efficiently expressed in cells, is easy to separate and purify, and has higher activity.

Description

Polypeptide with antibacterial and anti-inflammatory activity and application thereof

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a polypeptide with antibacterial and anti-inflammatory activities and application thereof.

Background

The antibacterial polypeptide is a micromolecular defense polypeptide synthesized by organisms, has broad-spectrum bactericidal action, has stronger killing action on gram-negative bacteria and gram-positive bacteria, has good killing action on certain fungi, mycoplasma and particularly drug-resistant bacteria, and is an important member of an animal innate immune system. Common antibiotics play a role by blocking the biosynthesis of biological macromolecules, and antibacterial polypeptides can perforate a bacterial cell membrane to form an ionic channel, so that the structure of the bacterial cell membrane is damaged, a large amount of intracellular water-soluble substances are leaked out, and finally bacteria die. With the widespread use of conventional antibiotics, many pathogenic bacteria have developed resistance to them, and thus, active polypeptides have significant advantages in terms of antibacterial activity.

In the prior art, scientists have discovered and isolated thousands of polypeptides having antibacterial activity from bacteria, fungi, higher plants, invertebrates, vertebrates, and even humans. For example, the soybean bioactive polypeptide Lunasin is a natural polypeptide separated and identified from soybeans, has the molecular weight of about 5kDa and has 43 amino acid residues in the whole length, and can block mitosis and cause chromosome breakage by transfecting mammalian cells to finally cause apoptosis.

However, the naturally separated active polypeptide Lunasin has the defects of low content and low activity, is not beneficial to separation and purification and limits the application.

Disclosure of Invention

The invention provides a polypeptide with antibacterial and anti-inflammatory activity and application thereof, wherein the structure of the existing polypeptide Lunasin is improved, the polypeptide is efficiently expressed in cells, is easy to separate and purify and has higher activity.

The first purpose of the invention is to provide a polypeptide with antibacterial and anti-inflammatory activity, wherein the nucleotide sequence of the polypeptide is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.

The second object of the present invention is to provide a recombinant vector prepared by using the polypeptide of claim 1, which is obtained by inserting the nucleotide sequence of SEQ ID No.1 into plasmid pET-32a (+).

It is a third object of the present invention to provide a transformant comprising the recombinant vector of claim 2.

A fourth object of the present invention is to provide a use of the polypeptide of claim 1 for inhibiting inflammatory response induced by LPS.

The fifth object of the present invention is to provide a use of the polypeptide of claim 1 against propionibacterium acnes.

Compared with the prior art, the polypeptide with antibacterial and anti-inflammatory activities and the application thereof provided by the invention have the following beneficial effects:

the G-Lunasin is an improvement on the amino acid structure of the existing polypeptide Lunasin, and the G-Lunasin enters target cell nuclei by utilizing the action of the GRDGRD at the 32 th position to the 37 th position, so that the combination efficiency with the target cells is improved, and compared with the natural Lunasin, the G-Lunasin promotes high-efficiency expression and increases the biological activity. The H amino acid is added after GRDGRD, on the basis of not influencing the activity of the polypeptide, the binding efficiency of the polypeptide and an affinity Ni column is increased, and the separation and purification are facilitated.

Drawings

FIG. 1 is an electrophoretogram of a target gene fragment and a plasmid fragment after gel recovery;

wherein, Lane M is DNAaker, Lane 1 is target gene fragment, Lane 2 is plasmid fragment;

FIG. 2 shows the Xba I and Pst I double restriction enzyme identification map of the positive clone;

wherein, Lane M is DNAmaker, Lane 1 is empty plasmid after double enzyme digestion, Lane 2 is recombinant plasmid after double enzyme digestion;

FIG. 3 shows the result of SDS-PAGE electrophoresis;

wherein, Lane M is DNAaker, and Lane 1 is G-Lunasin polypeptide expression product.

Detailed Description

The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The polypeptide (hereinafter referred to as G-Lunasin) with antibacterial and anti-inflammatory activities provided by the invention has the nucleotide sequence shown in SEQ ID No.1 and the amino acid sequence shown in SEQ ID No.2, improves the amino acid structure of the active polypeptide Lunasin, adds amino acid residues in the Lunasin amino acid sequence shown in SEQ ID No.3, increases the activity of the original Lunasin and the expression efficiency in cells, and reduces the difficulty of separation and purification of the Lunasin.

Based on the same invention concept, the invention also provides a preparation method of G-Lunasin, which comprises the following steps:

s1, construction of recombinant vector

An Nde I restriction site is added to one end of the nucleotide sequence shown in SEQ ID NO.2, an Xho I restriction site is added to the other end of the nucleotide sequence to obtain a target gene, and the target gene is inserted into a pUC57 plasmid for storage to obtain a recombinant plasmid pUC 57-G-Lunasin.

Carrying out double digestion on the recombinant plasmid pUC57-G-Lunasin by Nde I and Xho I, wherein the digestion system is shown in Table 1, the digestion conditions are 37 ℃ and 2 hours, and the target gene fragment with the G-Lunasin nucleotide sequence is recovered by gel;

the plasmid pET-32a (+) is subjected to double enzyme digestion by Nde I and Xho I, the enzyme digestion system is shown in the table 2, the enzyme digestion condition is 37 ℃, 2 hours, and plasmid fragments with longer sequences are gel recovered;

FIG. 1 is an electrophoretogram of a target gene fragment and a plasmid fragment after gel recovery, wherein Lane M is DNAmaker, Lane 1 is the target gene fragment, Lane 2 is the plasmid fragment, the band is clear, and no impurity band is present.

The target gene fragment and the plasmid fragment were ligated overnight with T4 ligase, and the ligation system was as shown in Table 3, to construct a recombinant vector pUC57-G-Lunasin-pET-32 a.

The plasmid pET-32a (+) is widely applied to expression of fusion proteins and polypeptides, and is provided with a histidine tag and the like, so that separation and purification of downstream polypeptide proteins are facilitated.

TABLE 1 restriction enzyme digestion System of recombinant plasmid pUC57-G-Lunasin

TABLE 2 digestion System for plasmid pET-32a (+)

Plasmid pET-32a (+) (30 ng/. mu.L)	40μL
		Nde I	3μL
Xho I	2μL
		10×buffer	5μL
General System	50μL

TABLE 3 connection System

Name of reagent	Amount of the composition used
		Target gene fragment (30ng)	6μL
10×T4DNA ligase Buffer	2μL
		Plasmid fragment (50ng)	5μL
T4DNA ligase(5U/μL)	1μL
		ddH₂O	20μL

S2, transforming and preparing the expression engineering bacteria

Transforming a recombinant vector pUC57-G-Lunasin-pET-32a into an escherichia coli DH5 alpha DE3 competent cell, selecting a positive transformant as an expression engineering bacterium, and adopting a conventional transformation method to obtain the recombinant vector pUC57-G-Lunasin-pET-32 a. Screening for positive transformants was also performed according to the conventional method. The positive clone is selected and identified by Xba I and Pst I double enzyme digestion, the result is shown in figure 2, and the result shows that the recombinant vector is constructed correctly.

S3, polypeptide expression and purification

S31, inoculating the expression engineering bacteria into LB (Amp resistance) culture medium, performing shake culture at 37 ℃ and 220rpm overnight to obtain seed liquid, transferring the seed liquid into fresh LB (Amp resistance) culture medium according to the volume ratio of 2% the next day, and continuously culturing until OD is reached₆₀₀When the concentration is 0.6, adding IPTG (isopropyl-beta-thiogalactoside) to enable the final concentration of the IPTG to be 0.1mM, and carrying out shake culture at 30 ℃ and 220rpm for 4h to obtain polypeptide expression bacterial liquid; taking 1mL of polypeptide expression bacterial liquid, centrifuging at 5000rpm and 4 ℃ for 5min, and collecting thalli; after IPTG induction expression, the expression product is detected by SDS-PAGE electrophoresis, and the result is shown in figure 3, wherein a lane M is DNAmaker, a lane 1 is G-Lunasin expression product, and a clear band is visible near 6 KDa.

S32, 1g of the cells were collected, and 5% by mass of the cells of a Bingding Buffer solution A (20mM phosphate, 0.5M sodium chloride, 5mM imidazole, pH7.4) and PMSF were added. PMSF was prepared as a 100mM stock solution in absolute ethanol, working concentration of PMSF was 1 mM. Adding the Bingding Buffer solution A and PMSF to resuspend the cell sediment, adding lysozyme (working concentration is 0.3mg/mL), mixing evenly, and standing on ice for 30 min. Breaking cells by ultrasonic on ice at 400W, stopping ultrasonic for 5s, and repeating for 40 times. Adding 10% TritonX-100 to final concentration of 0.05%, mixing, and standing on ice for 15 min. Centrifuging at 12000rmp and 4 deg.C for 20min, collecting supernatant, filtering with 0.22 μm membrane to obtain filtrate, and storing on ice for use.

S33, Ni Sepharose resin is loaded on a suitable chromatographic column, separated with an eluent of 10 times the volume of the filtrate, and passed through a Bingding Buffer solution A (containing 50mmol of NaH)₂PO₄5mmol of imidazole, 500mmol of NaCl in the presence of double distilled water at pH7.4, solution B (containing 50mmol of NaH)₂PO₄20mmol of imidazole, 500mmol of NaCl, the solvent is double distilled water, the pH is 7.4), and a solution C (containing 50mmol of NaH)₂PO₄60mmol of imidazole, 500mmol of NaCl, the solvent being double distilled water, pH7.4), solution D (containing 50mmol of NaH)₂PO₄100mmol of imidazole and 300mmol of NaCl, the solvent is double distilled water, the pH is 7.4), the eluates are respectively collected at the flow rate of 12-15mL/h, the purity of G-Lunasin in the eluent D is up to 98.3 percent and the content of G-Lunasin in the eluent D is up to 0.05G/mL, and the purity of the natural Lunasin extracted by the method is 90.3 percent and the content of G-Lunasin in the eluent D is 0.03G/mL.

First, G-Lunasin toxicity detection

Preparing 0, 10 mu M, 30 mu M and 60 mu M G-Lunasin solutions respectively, stimulating macrophage RAW264.7, adding medicine for 24h, and then detecting and stimulating macrophage activity by MTT, wherein the results are 100%, 104%, 110% and 98%, respectively, and the results show that the 0-60 mu M G-Lunasin polypeptide solution is non-toxic to macrophage viruses.

Second, Effect of G-Lunasin on NO concentration in cell culture Medium

Sterile water was used as a negative control, and the NO production in the NO cell culture medium was 2.4. mu.M after 24 h. LPS with the concentration of 1 mu g/mL is used as a positive control group to stimulate macrophage RAW264.7, and the NO yield in the NO cell culture medium is detected to be 5.5 mu M after 24 hours. Then, 10. mu.M, 30. mu.M, 60. mu.M G-Lunasin solution and 100. mu.M natural Lunasin solution were added to 4 LPS-stimulated cell culture substrates, respectively, and after 24 hours, the concentrations of NO in the cell culture substrates were detected to be 4.5. mu.M, 4.3. mu.M, 4.1. mu.M and 4.8. mu.M, respectively. The G-Lunasin solution can obviously reduce the NO increase phenomenon caused by LPS, has good biological activity, can effectively inhibit inflammatory reaction caused by LPS, and has more obvious effect than the natural Lunasin solution.

Third, G-Lunasin antibacterial effect

After a rat acne model was successfully constructed by intradermally injecting 50 μ L of a suspension of the Acetobacter acnes in the center of the auricle of the rat, group A of the acne model was not administered with any treatment; the skin lesion of the group B is externally treated by applying a 0.1 percent clindamycin solution; c group skin lesions are treated by external application of G-Lunasin solution with the mass fraction of 0.05%. The control group was administered without injection of the central auricle of rats with a suspension of Acidocella acnes.

The skin of the injection part of the three groups of rats begins to thicken and gradually increases at the time of 24h of injection, and acne-like skin lesions such as acne, papule, pustule and the like appear on the 5 th day, and the skin is red and hard; B. after 2 weeks of treatment in group C, the abscesses had substantially subsided, the thickness was reduced and the color tended to be normal. Skin tissues of injection sites of the right ears of rats in each group are taken, HE staining is carried out, the skin epidermis of the left ear of the rat in the control group is thinner, hair follicles can be seen, and the junction of the epidermis and the dermis is clear; the epidermis of the group A is over-keratinized, the area of hair follicles is enlarged, and inflammatory cells are subjected to diffuse infiltration; B. the keratinization of the hair follicle in group C was significantly reduced, and a small amount of inflammatory cells were visible in the dermis. The G-Lunasin solution has good antibacterial effect. B. No toxic side effects were observed in group C rats.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Sequence listing

<110> Henan institute of engineering

<120> polypeptide with antibacterial and anti-inflammatory activity and application thereof

<160>3

<170>PatentIn version 3.5

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<213> Artificial sequence

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tccaaatggc agcaccagca agacagctgc cgcaagcagc tccagggggt gaacctcacg 60

ccttgcgaga agcacatcat ggagaagatc caaggccgcg gcggccgcgg ccacgatgac 120

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Ser Lys Trp Gln His Gln Gln Asp Ser Cys Arg Lys Gln Leu Gln Gly

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Val Asn Leu Thr Pro Cys Glu Lys His Ile Met Glu Lys Ile Gln Gly

20 25 30

Arg Asp Gly Arg Asp His Asp Asp Asp Asp Asp Asp Asp Asp Asp

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Ser Lys Trp Gln His Gln Gln Asp Ser Cys Arg Lys Gln Leu Gln Gly

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Val Asn Leu Thr Pro Cys Glu Lys His Ile Met Glu Lys Ile Gln Gly

20 25 30

Arg Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp

35 40

Claims

1. A polypeptide with antibacterial and anti-inflammatory activity is characterized in that the nucleotide sequence of the polypeptide is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2; the polypeptide is used for preparing an inhibitor of LPS inflammatory reaction or an antibacterial agent of propionibacterium acnes.

2. A recombinant vector produced by using the polypeptide of claim 1, wherein the recombinant vector is obtained by inserting the nucleotide sequence of SEQ ID NO.1 into a plasmid pET-32a (+).

3. A transformant comprising the recombinant vector of claim 2.