CN114381456B

CN114381456B - Artificially synthesized nano silver synthetic protein gene, expressed protein and application thereof

Info

Publication number: CN114381456B
Application number: CN202111665678.8A
Authority: CN
Inventors: 时号; 言行; 李青飞; 聂新玲; 高凤; 王士岩; 李相前
Original assignee: Huaiyin Institute of Technology
Current assignee: Shenzhen Morning Intellectual Property Operations Co ltd
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2023-09-22
Anticipated expiration: 2041-12-31
Also published as: CN114381456A

Abstract

The invention discloses an artificially synthesized nano silver synthetic protein gene, an expressed protein and application thereof, and the artificially designed nano silver synthetic protein with certain reducibility is supported by the related technology of protein engineering according to the common characteristics of ferritin family protein sequences. The DNA sequence of the protein is shown as SEQ NO. 1, and the amino acid sequence is shown as SEQ NO. 2. The protein can efficiently synthesize nano silver, has high synthesis efficiency, and the synthesized nano silver solution is brown, and has good broad-spectrum antibacterial property and remarkable antibacterial effect.

Description

Artificially synthesized nano silver synthetic protein gene, expressed protein and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and relates to a nano silver synthetic protein, in particular to an artificially synthesized nano silver synthetic protein gene, and an expressed protein and application thereof.

Background

The nano silver is a metal simple substance with the particle size reaching the nano level, has tiny particles and large specific surface area, and has super-strong permeability. The nano silver changes the cell membrane structure by releasing anions, so that cell fluid leaks; can also react with the sulfhydryl groups in important enzymes in the cell interior to prevent the cell from dividing; it also prevents DNA replication and thus causes apoptosis. Because of its physical antibacterial ability, nano silver has broad-spectrum antibacterial property and extremely low drug resistance which are not possessed by the traditional inorganic antibacterial agents.

Currently, the mainstream nano-silver synthesis methods are classified into three categories: 1. physical methods, using mechanical grinding or laser ablation of bulk silver material to obtain nano silver powder. The product obtained by synthesizing nano silver by a physical method is easy to agglomerate, and a plurality of steps which are difficult to control exist in the synthesis process, so that the energy consumption is high and the requirement on equipment is high. 2. In the chemical method, a reducing agent, a protective agent and a dispersing agent are added into silver salt solution, and silver salt is reduced into silver simple substance under proper conditions. The process is simple, but more chemical reagents are used in the process, so that the environmental pollution is large, and the subsequent waste liquid treatment is difficult. 3. The biological method is used for preparing nano silver through biological functions inside and outside biological cells. The nano silver is prepared by utilizing certain organisms in the nature, such as extracting solutions with strong reducibility, such as methanogen or fungi and active ingredients in plants, the preparation condition is mild, any chemical reagent which is unfavorable for environmental protection is not needed, biological resources are fully utilized, and the sustainable development green environment-friendly effect can be achieved.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide an artificially synthesized nano silver synthetic protein gene, and an expressed protein and application thereof. The protein expressed by the artificial synthetic protein gene lock belongs to the ferritin family protein and can be used for efficiently synthesizing nano silver.

The invention is realized by the following technical scheme:

an artificially synthesized nano silver synthetic protein gene, the DNA sequence of which is shown in SEQ NO. 1.

The invention also provides the nano silver synthetic protein obtained by coding the artificially synthesized nano silver synthetic protein gene.

Further, the nano silver synthetic protein is a protein of the following (1) or (2):

(1) A protein having an amino acid sequence as shown in SEQ NO. 2;

(2) The protein with nano silver synthesis capability is formed by substituting, deleting and adding one or more amino acid residues in the amino acid sequence shown as SEQ NO. 2.

The invention also provides a recombinant vector, which consists of an empty vector and a target gene inserted into the empty vector, wherein the target gene is a gene with a DNA sequence shown as SEQ NO. 1.

Further, the empty vector is pET-28a.

Furthermore, the invention also provides a recombinant bacterium, wherein the recombinant bacterium has a gene with a DNA sequence shown as SEQ NO. 1.

The invention further improves the scheme as follows:

the application of the nano silver synthetic protein in synthesizing nano silver.

Further, when synthesizing nano silver, the concentration of the silver nitrate substrate is 5-50mM, the synthesis reaction temperature is 20-70 ℃, the pH is 11, and the reaction time is 12-24h.

Compared with the prior art, the invention has the beneficial effects that:

1. according to the common characteristics of methanogen iron sulfur protein family, by means of the related technical support of protein engineering and combining the related parameters of reducibility, water solubility and the like of various amino acids, various possible conditions of combining with silver ions are simulated through molecular pairing, and the amino acid sequence shown as SEQ NO. 2 is designed and screened. The protein can synthesize nano silver under alkaline condition without other reagents. The synthesis conditions are simple, the equipment requirements are low, and the method is environment-friendly.

2. When the artificially designed iron-sulfur protein family protein is used for efficiently synthesizing nano silver, the pH range is 10-11, the temperature range is 20-70 ℃ and the synthesis time is 12-24h. The obtained nano silver solution is brown, has stable property and has antibacterial effect after being stored for one month at room temperature.

3. The nano silver synthesized by the protein has good antibacterial effect on staphylococcus aureus, escherichia coli, bacillus subtilis and yeast, and has wide application prospect and industrial value.

Drawings

FIG. 1 is an electrophoresis diagram of an artificially designed protein of the iron-sulfur protein family Mcy SDS-PAGE;

FIG. 2 is a full-wavelength scan of a 5-fold dilution of a synthetic nanosilver solution;

FIG. 3 shows the results of a plate bacteriostasis experiment using the protein synthesized nanosilver of the present invention, wherein a is yeast, b is Staphylococcus aureus, and c is Escherichia coli.

Detailed Description

The invention will be further illustrated with reference to specific examples.

Materials, reagents and the like used in the following examples were obtained commercially unless otherwise specified.

EXAMPLE 1 preparation of artificially designed genes for the iron-sulfur protein family proteins

Summarizing the preference and structural commonality of the methanogen ferritin family protein amino acid through database comparison analysis, and designing an amino acid sequence framework by means of protein engineering technology; simulating the three-dimensional structure of the amino acid sequence skeleton by using alpha fold 2; using AutoDock to simulate the interaction condition of silver ions and proteins, optimizing an amino acid skeleton, and preferentially selecting amino acids with higher activity such as aspartic acid, histidine, serine and the like as electron donors in nano silver synthesis; the high-frequency amino acid in the heat-resistant proteins such as proline, isoleucine and arginine are matched for supplementing, so that the structural strength of the protein is improved; protein solubility is also predicted taking into account protein solubility. The amino acid sequence shown as SEQ NO. 2 is obtained; and designing a DNA sequence shown as SEQ NO. 1 according to codon preference of an expression host, and artificially synthesizing the gene fragment.

Example 2 construction and verification of recombinant cloning and expression vectors.

The synthesized target gene fragment (prepared in example 1) and pET-28a (Novagen) were digested with XbaI and XhoI, respectively, and the digested PCR and large fragment of the vector were recovered by agarose electrophoresis. The target fragment after the rubber cutting recovery was ligated with the vector by adding 1. Mu.L of 10 XLigase Buffer and 1. Mu.L of Ligase overnight at 16 ℃. Coli DH 5. Alpha. Was transformed with the ligation reaction product, and then plated on a petri dish containing 100. Mu.g/mL Kana (Kana penicillin), and incubated at 37℃for 10-15 h.

A plurality of single colonies were picked from the transformation plate and plasmids were extracted using the plasmid miniprep kit of BIOMIGA. The obtained plasmid was verified by double cleavage and the obtained recombinant plasmid was sequenced. Sequencing results show that the cloned target fragment (with the nucleotide length of 432 bp) is inserted into the pET-28a vector, so that recombinant clone and an expression vector pET-28 a-Mcy 11 are obtained, the DNA sequence of the expressed protein gene is shown as SEQ NO. 1, the amino acid sequence of the expressed protein is shown as SEQ NO. 2, and the protein is named as Tp Mcy11.

EXAMPLE 3 expression and purification of recombinant nanosilver synthetic protein Tp Mcy11

Recombinant cloning and expression vector pET-28 a-Mcy 11 (prepared in example 2) are subjected to heat shock transformation to host bacteria E.coli BL21 (DE 3) (Novagen) to obtain recombinant bacteria containing recombinant plasmids. Single colony recombinant bacteria were inoculated in 5 mL Luria-Bertani broth (LB) medium containing 100. Mu.g/mL of kanapecillin, and shake cultured at 200 rpm at 37℃for 4h. The 4. 4 mL strain is inoculated into a 2000 mL shake flask containing 800 mL culture medium, cultured under shaking at 37℃and 200 rpm, when the absorbance reaches 0.4-0.6, 800. Mu.L of 0.1M IPTG is added, and induced to express 15 h at 22℃and 150 rpm. The culture broth was centrifuged at 6000 rpm for 10 min at 4℃with a high-speed refrigerated centrifuge, and the cells were collected. The cells were recovered by washing with 50 mL ultra pure water and centrifuging at 6000 rpm for 10 min at 4℃followed by 20: 20 mL Binging Buffer resuspension (0.5M NaCl,20mM Tris-HCl,5mM Imidazole,pH 7.4), disrupting the bacterial cells with an ultrasonic disrupter in an ice-water bath and centrifuging at 10000 rpm for 50 min at 4℃to obtain a crude extract of recombinant nano silver synthetic protein Tp Mcy11.

The crude extract was purified using a Ni-NTA affinity column (see His-Band kit, novagen). The purity of the purified protein was identified and the molecular weight was determined by SDS-PAGE, and the results are shown in FIG. 1,1 for 200 mM imidazole eluted purified Mcy protein. The molecular weight is about 16 kDa, which is close to the theoretical value.

Example 4 Synthesis of nanosilver Using recombinant nanosilver Synthesis protein Tp Mcy11

The purified protein solution obtained in example 3 was dialyzed to remove imidazole and NaCl, concentrated to 15mg/ml by ultrafiltration, and concentrated according to the silver nitrate concentration (mM): the final protein concentration (μg/ml) =1:2 ratio was prepared by preparing 30ml of the reaction solution, adjusting the pH to 11 with sodium hydroxide, and shaking the solution for 20 hours at 30 ℃ with a shaking table at 150rpm to obtain a dark brown nano silver solution, the full wavelength scan of which is shown in fig. 2. The nano silver solution generally shows an absorption peak at a full wavelength of 410mm-440mm, and the higher the nano silver concentration, the higher the absorption peak in a certain concentration range. In the case of nanoparticles, the absorption wavelength of the nanomaterial moves in the short wave direction due to quantum size effect as the particle size decreases; the smaller the particle diameter of the synthesized nano silver particles, the more the peak is shifted in the 400mm direction. The full-wavelength scanning result of the nano silver particles synthesized by the protein shows that the obtained nano silver particles have uniform particle diameter and smaller particle diameter, and the particle diameter distribution is about 20mm through dynamic light diffraction analysis.

Example 5 experiment of Nano silver antibacterial Ring

Preparation of pathogen plates: 2ml of an overnight culture medium of pathogenic bacteria (Staphylococcus aureus ATCC6538, escherichia coli ATCC25922, torulopsis globosa) was added to 100ml of LB medium containing 2% nutrient agar, and the mixture was poured into a dish. 6. Mu.l of 50. Mu.g/ml ampicillin or kanamycin sulfate, the supernatant of the nano silver synthesis solution without adding protein, the protein solution with sodium chloride removed by dialysis, and the nano silver solution synthesized by Tp Mcy11 were adsorbed on each filter paper sheet. Culturing in an incubator at 37 ℃ for 24 hours. The situation of the inhibition zone is found: the supernatant of the nano silver synthetic solution without protein and the protein solution have no antibacterial effect, and the supernatant of the nano silver synthetic solution with protein has obvious antibacterial circle, which proves that the protein can synthesize nano silver, and the nano silver has a certain antibacterial effect on fungi (candida globosa), gram positive bacteria (staphylococcus aureus ATCC 6538) and gram negative bacteria (escherichia coli ATCC 25922).

Sequence listing

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<120> an artificially synthesized nano silver synthetic protein gene, and expressed protein and application thereof

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Glu Val Asn Gln Gln Thr Glu Asp Ile Asp Arg Asp Ile Arg Val Lys

1 5 10 15

Tyr Gln Arg Ile Lys Thr Gly Phe Ile Ile Ile Pro Met Arg Lys Glu

20 25 30

Gln Glu Val Met Lys Ile Leu Ser Thr Gln Phe Thr Val Pro Glu Tyr

35 40 45

His Gly Thr Val Thr Glu Ala Leu Glu Val Asn Glu Lys Leu Ile Asp

50 55 60

Arg Ala Gly Lys Glu Ser Gln Gln Val Thr Pro Asp Leu Asp Asp Thr

65 70 75 80

Val Pro Phe Met Ala Glu Glu Met Lys Gly Leu Pro Gly Leu Arg Val

85 90 95

Ile Asn Glu Ile Asp Asp Ile Asp Leu Asn Asp Ala Ile Glu Cys Gly

100 105 110

Lys Gly Arg Leu Gly Asn Met Ile Glu Lys Leu Glu Ala Ile Gln Thr

115 120 125

Asp Glu Ala His Val His Lys Leu Glu Leu Phe Ile Arg Glu Thr Leu

130 135 140

Claims

1. An artificially synthesized nano silver synthetic protein is characterized by being obtained by encoding a gene shown in SEQ NO. 1.

2. A recombinant vector comprising an empty vector and a target gene inserted into the empty vector, wherein the target gene is the gene according to claim 1.

3. A recombinant vector according to claim 2, wherein: the empty vector is pET-28a.

4. A recombinant bacterium comprising the gene of claim 1.

5. Use of the nano-silver synthesis protein according to claim 1 for synthesizing nano-silver.

6. The use of the nano-silver synthetic protein according to claim 5, wherein: when synthesizing nano silver, the concentration of the silver nitrate substrate is 5-50mM, the synthesis reaction temperature is 20-70 ℃, the pH is 11, and the reaction time is 12-24h.