CN113444721B - A kind of artificially modified gene, SNA15 protein and its method for efficiently synthesizing nano silver - Google Patents

Abstract

The invention discloses an artificial modified gene, SNA15protein and a method for efficiently synthesizing nano silver by using the same, wherein the nucleotide sequence of the gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein is shown as SEQ ID NO.2. The invention discloses a nucleic acid sequence corresponding to SNA15protein amino acid which can be used for efficiently synthesizing nano silver after artificial transformation for the first time. The biological nano silver solution prepared by the invention is brown, has stable property and particle size distribution of 2-25nm, and the synthesized nano silver surface is coated with protein participating in synthesizing nano silver, so that the biological nano silver solution has good biological function, and antibacterial experiments prove that the nano silver has broad-spectrum antibacterial effect and remarkable antibacterial effect.

Description

A kind of artificially modified gene, SNA15 protein and its method for efficiently synthesizing nano silver

technical field

The invention relates to genetic engineering and protein engineering, in particular to an artificially modified gene, SNA15 protein and a method for efficiently synthesizing nano silver.

Background technique

Silver has been used as an antibacterial agent since ancient times, but with the development of technology and the emergence of antibiotics, the utilization rate of silver in sterilization has declined. However, due to the abuse of antibiotics, the time from the development of new drug categories to the detection of bacterial resistance to new drugs is gradually shortening. Taking penicillin as an example, most of the third and fourth generations of penicillin are currently used.

The antibacterial mechanism of nano-silver is different from that of antibiotics. Nano-silver has a broad spectrum of antibacterial properties. The smaller the particle size of nano-silver, the stronger its bactericidal performance. And a type of antibiotic can only inhibit a certain type of bacteria, because the antibacterial effect of nano-silver is to cause the death of bacteria through direct exposure. Due to its special nano-size, it leads to nano-perforation of bacteria, so that nano-silver is inserted into the cell membrane or cell wall of bacteria, causing its structure to be destroyed, resulting in the outflow of bacterial intracellular components, causing bacterial death. When nano-silver enters the bacterial cytoplasmic membrane, it will combine with thiol-containing LPS, weakening its interaction and increasing the permeability of the cell membrane. At the same time, nano-silver is easy to combine with oxygen and promotes the dissociation of _O2 to generate ROS. The smaller the particle size of nano-silver, the stronger its ability to generate ROS. Nanosilver invaded into the cell will cross-link with the DNA and protein in the cell, resulting in the loss of oxidative damage repair genes and oxidative damage proteins, and the condensation of free DNA molecules, affecting the normal division of cells and the expression of functions.

Using nano-silver as an antibacterial agent plays an important role in water purification, food preservation, aquaculture, agricultural production and biomedical applications. At the same time, nano-silver is also used in high-tech fields such as sensors in the form of nano-silver paste. With the rapid development of nanomaterial technology, the synthesis of nanoparticles has become more and more convenient, and nanoparticles have been mass-produced and widely used in daily life. Since the surface of nano-silver has the function of plasmon resonance enhancement, and different shapes, different particle sizes of nano-silver and different excitation wavelengths will affect the fluorescence enhancement effect of nano-silver. Taking the spherical silver nanoparticles synthesized by this protein as an example, the larger the particle size, the more the infrared shift of its surface plasmon resonance peak, and the surface plasmon resonance effect gradually changes from absorption to scattering. At the same time, nano-silver has a surface-enhanced Raman scattering effect, which can enhance the Raman signal of the substance to be tested, and has the characteristics of realizing single-molecule detection. It can effectively realize the application of composite probes in the detection of targeted SERS imaging of cancer cells, on-site detection of toxic and harmful substances in aqueous solutions, and detection of tumor cell proliferation. It is precisely because of the surface effect and quantum size of nano-silver that nano-silver plays an important role in the field of biomedicine, surface-enhanced Raman effect, and microelectronics. For example, combined application of topical recombinant human basic fibroblast growth factor and nano-silver antibacterial hydrogel dressing can reduce local swelling, relieve postoperative pain, promote wound recovery of open ankle fractures, relieve inflammation and oxidative stress, and effectively reduce the incidence of postoperative incision complications. Nano-silver antibacterial gel can prevent the occurrence of radiation dermatitis and reduce the severity of radiation dermatitis, reduce the treatment interruption caused by skin reactions, and improve the treatment effect.

There are currently three main methods for synthesizing nano-silver: the first is a simple and direct physical method, but the physical method has high requirements for equipment, which brings high costs and serious equipment loss, but the size of the nano-silver particles produced can meet people's expectations, and the production cycle is short; the second is chemical synthesis. This method brings environmental pollution and the use of toxic reagents, and chemical reduction is not conducive to the use of biomedical direction due to the increase in toxicity of irritating chemicals adsorbed on the surface of the synthesized silver nanoparticles. Compared with the other two methods, the method has a series of advantages such as mild reaction, low production cost, green environmental protection and no pollution, and can complete the preparation of nano-silver without additives such as reducing agents in the reaction process. With the popularization of green and environmental protection concepts, the biological method of using biological systems to synthesize nano-silver particles has attracted much attention. Under the protection of biomolecules, the stability and dispersibility of biological nano silver have been greatly improved. It has good biocompatibility, special physical and chemical properties, excellent antibacterial, anti-inflammatory effects and low biological toxicity. It has unique advantages and a pivotal position in the field of biomedical materials. Therefore, it is of great significance for the preparation of more efficient and high-yield antibacterial silver nano-products to study the optimal synthesis conditions of biosynthetic nano-silver particle size and to understand the synthesis mechanism of nano-silver. In the prior art, CN110804089 proposes a nano-silver synthetic protein derived from Bacillus lysinus globosa and its application. Although it discloses a protein for synthesizing nano-silver, the nano-silver efficiency of its protein synthesis is low, and the synthetic nano-silver has a poor dispersion effect and a large particle size. In order to obtain a more efficient protein for synthesizing nano-silver, the protein is transformed.

Contents of the invention

Purpose of the invention: Aiming at the problems existing in the prior art, the present invention provides a gene encoding and synthesizing nano-silver protein after artificial transformation. The protein encoded by the gene of the present invention can efficiently synthesize nano-silver. At the same time, the synthesis of nano-silver by the protein obtained by prokaryotic expression and purification belongs to non-enzyme-catalyzed synthesis. It is of great significance in the field of synthesizing nano-silver with a single non-enzyme-catalyzed protein.

The invention also provides proteins, carriers, engineering bacteria and applications capable of efficiently synthesizing nano silver with biological activity.

Technical solution: In order to achieve the above object, the present invention provides a gene encoding a synthetic nano-silver protein, the nucleotide sequence of which is shown in SEQ ID NO.1.

Wherein, the gene uses the nucleotide of the known nano silver protein as a template (SEQ ID NO.2 in CN110804089; SEQ ID NO.5 in the sequence listing of the present invention), and performs spatial simulation on the synthetic nano silver protein structure encoded by the known nucleotide, analyzes the functions and effects of each structural domain, performs protein structural domain rearrangement, and screens to obtain a structural domain that can efficiently synthesize nano silver. Using the gene sequence encoding the rearranged protein after the rearrangement as a template, the sequence obtained by site-directed mutagenesis is encoded and efficiently synthesized as shown in SEQ ID NO 1 The gene of nanosilver protein. The target gene is connected with the vector to obtain a recombinant vector, and the recombinant vector is introduced into a prokaryotic expression host for expression and purification.

The amino acid sequence of the protein encoded by the gene of the present invention capable of efficiently synthesizing nano silver with biological activity is shown in SEQ ID NO.2.

The recombinant vector of the present invention includes the gene encoding the synthetic nano-silver protein.

The engineering bacterium of the present invention includes the gene encoding the synthetic nano-silver protein or the synthetic nano-silver protein or a recombinant vector.

The application of the gene encoding the synthetic nano-silver protein described in the present invention in preparing nano-silver through a biological method.

The application of the synthetic nano-silver protein of the present invention in preparing nano-silver through a biological method.

Wherein, in the application, silver nitrate is used as the substrate concentration of 14-30mM, the reaction temperature range of synthesizing nano-silver is 20-50°C, the rotation speed is 100-250rpm, the pH is 9-13, and the reaction time is 16-24h.

Wherein, the nano-silver prepared in the application is spherical nano-silver, and the particle size distribution is in the range of 2-25nm. Moreover, the synthesis conditions of nano-silver are controllable each time, and the particle size of the synthesized nano-silver is controllable. The protein coated on the surface of the nano-silver synthesized by the biological method has good biological activity.

Further, the nano-silver prepared in the application is spherical nano-silver with a particle size distribution of 2-25nm, and its surface is coated with proteins involved in the synthesis of nano-silver. After scanning electron microscopy (SEM) characterization, it was found that nano-silver with a particle size of less than 10nm was in an agglomerated state. According to the SEM characterization results, the particle size of the agglomerated nano-silver was around 80nm. The reason was that the particle size of the nano-particle decreased and the proportion of its surface atoms increased, resulting in insufficient surface coordination number and high surface energy, so that the nano-silver was in a highly activated state, so that the nano-silver with a small particle size would appear in an agglomerated state. After the sample was dispersed by ultrasonic waves, TEM was used to characterize it.

The invention utilizes genetic engineering technology to modify the gene encoding and synthesizing the nano-silver protein. The protein encoded by the obtained engineering gene can efficiently synthesize nano silver, the protein has a high expression content in the prokaryotic expression system, and the protein is easy to be purified from the prokaryotic expression system by protein purification, and the protein can stably synthesize a large amount of nano silver under certain conditions. The present invention proposes that protein sequences and nucleic acid sequences of nano-silver can be synthesized in large quantities after genetic engineering and protein engineering transformation, and the protein can be prepared by a biological method under normal temperature and isothermal conditions. At the same time, the nano-silver synthesized by the biological method has been proved by antibacterial experiments that the nano-silver has a broad-spectrum antibacterial effect and the antibacterial effect is remarkable. In addition, the protein of the present invention can be synthesized with nano-silver under controllable conditions and particle size range, and is not affected by other factors. As long as it is within the pH range, nano-silver can be synthesized without a particularly high temperature, and the particle size of nano-silver measured by DLS is also within the synthesis range, and a large number of nano-silver particles can be industrially produced.

The present invention successfully amplifies its corresponding DNA, and successfully constructs a recombinant vector, and obtains a prokaryotic expression host or engineering bacteria carrying the recombinant vector. Since the prokaryotic expression system has many advantages such as simple, rapid, economical and suitable for large-scale industrial production processes, the preparation of nano-silver by using a single protein in the biological method has the prototype of industrial scale application, and has broad application prospects and industrial value.

The present invention uses genetic engineering and protein engineering techniques to artificially transform the protein, which can reduce the substrate silver nitrate to nano-silver, which is beneficial to revealing the synthesis mechanism of nano-silver, and is also beneficial to the industrial production and application of biosynthetic nano-silver. At the same time, the antibacterial ability of the biologically synthesized nano-silver is explored with Staphylococcus aureus, Escherichia coli, Penicillium, and Saccharomyces cerevisiae.

Beneficial effect: compared with the prior art, the present invention has the following advantages:

1. After genetic engineering technology, protein engineering technology, and artificial transformation, it can efficiently synthesize nano-silver artificial sequences and artificial proteins with biological activity. The gene uses the nucleotide sequence of the known nano-silver protein as a template (SEQ ID NO.2 in CN110804089) to carry out space simulation, analyze the functions and effects of each structural domain, screen and obtain nano-silver high-efficiency synthesis domains, and use site-directed mutagenesis for mutation to obtain a new artificial sequence. The protein encoded by the gene can efficiently synthesize nano-silver, and can play a role in reducing the agglomeration of small-sized nano-silver, and is used as a protective agent and a dispersant in the biological method. The gene encoding the protein is finally named SNA15, and its sequence is shown in SEQ ID NO 1. The amino acid sequence of the protein named SNA15protein is shown in SEQ ID NO.2. The present invention first adopts protein engineering technology to perform protein structure simulation analysis on the protein sequence, then rearranges the protein domains, then uses genetic engineering technology to perform site-directed mutagenesis at multiple sites of its corresponding DNA sequence, and designs PCR reaction conditions. The DNA sequence carrying the mutation site is successfully obtained through PCR site-directed mutation. The protein encoded by the mutated sequence can efficiently synthesize nano-silver and has a good dispersion effect in aqueous solution. Therefore, the present invention also discloses for the first time the nucleic acid sequence corresponding to the amino acid of SNA15 protein that can efficiently synthesize nano-silver after artificial transformation.

2. The SNA15 protein of the present invention has a pH range of 11 to 13 and a temperature range of 20 to 50°C. The synthetic efficiency of nano silver is relatively high, and the synthesis time is 16 to 24 hours. The obtained biological nano silver solution is brown and stable in nature, with a particle size distribution of 2 to 25 nm, and the surface of the synthesized nano silver is coated with protein, which has good biological activity and biological function.

3. The artificially modified protein of the present invention successfully synthesized nano silver, found the key functional region of the modified protein, and successfully amplified its corresponding DNA. After recombinant vector and prokaryotic expression, an engineering bacterium capable of efficiently synthesizing biological nano silver was obtained in large quantities. The expression level of the SNA15 protein is high, which is more than 10 times the expression level before artificial modification, and the protein is stable. The nano silver synthesized by SNA15 protein has good biological activity, controllable particle size, mild synthesis conditions, and SNA15 protein The synthesis of nano-silver belongs to non-enzyme-catalyzed nano-silver synthesis, which is different from the conventional enzyme-catalyzed synthesis of nano-silver. There is no mutation in the enzyme active center that makes it impossible to synthesize nano-silver. There is still no research on this aspect. The invention is conducive to revealing the synthesis mechanism of nano-silver prepared by using a single non-enzyme-catalyzed protein, and at the same time exploring the antibacterial ability of the nano-silver synthesized by the biological method to Staphylococcus aureus, Escherichia coli, Penicillium and Saccharomyces cerevisiae. It has extremely broad application prospects and industrial value.

Description of drawings

Fig. 1 is the agarose gel electrophoresis of target gene;

Fig. 2 is the agarose gel electrophoresis of pET-28a (+) recombinant plasmid double-enzyme digestion verification;

Fig. 3 is the SDS-PAGE electrophoresis pattern of the modified protein supernatant and the control group and the SDS-PAGE electrophoresis pattern of the original protein after affinity chromatography purification; wherein Fig. 3a is the SNA15 protein of the recombinant protein, and Fig. 3b is the protein before transformation (positive control);

Fig. 4 is the SDS-PAGE electrophoresis figure of anion chromatography purification;

Fig. 5 is nano-silver synthesis peak diagram and synthetic color; wherein Fig. 5a is the characteristic absorption peak diagram of nano-silver synthesized by different proteins under the same conditions, Fig. 5b is the color of nano-silver solution synthesized by different proteins under the same conditions, and Fig. 5c is the synthesis of nano-silver by SNA15 protein of recombinant protein at different temperatures;

Figure 6 is a SEM scanning electron microscope characterization diagram;

Figure 7 is a TEM characterization diagram;

Figure 8 shows the antibacterial effect of nano silver on Staphylococcus aureus, Escherichia coli, Saccharomyces cerevisiae and Penicillium.

Detailed ways

The present invention will be further described below in conjunction with the embodiments and the accompanying drawings.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1

Gene cloning, construction and verification of expression vectors

With the nucleotide of known nano silver protein as template (SEQ ID NO.2 in CN110804089; SEQ ID NO.5 in the sequence listing in the present invention), by carrying out spatial simulation to the synthetic nano silver protein structure of known nucleotide encoding, analyze each structural domain function and effect, carry out the structural domain rearrangement of protein, screen and obtain the structural domain that can efficiently carry out nano silver synthesis (screening method is the same as embodiment 2 and 3), with the gene sequence of coding rearrangement protein after rearrangement as template, design site-directed mutagenesis primer, primer cross Synthesized by Shanghai Jieli Biotechnology Co., Ltd., the site-directed mutation sequence is as follows:

SEQ ID NO. 3: F

GGAATTCCATATGGATCGAACTCGCCCAAAAGATAAGAAAGTAAAAGTGAAAAATTCAAAACTTTAGTTGTGACTTTCTCTAAAACATTAGATTCTTCAGATGGAAAC

SEQ ID NO.4: R

CCGCTCGAGAGCTACAGTAAATCCGTCAGCTGCTCTCTTCGTAGTTTCTGGTTTGAATACAGTTGCAAAGTCAGTTTTAAGATTAGCAATATGAGTTGAATCATCTTC

Site-directed mutation of the target gene by PCR. The PCR is a touch-down PCR, and the program design is as follows: pre-denaturation at 98°C for 5 minutes, denaturation at 98°C for 15s, initial annealing temperature at 65°C, annealing for 15s, a total of 15 cycles. The temperature was decreased by 1°C every other cycle, the annealing time was 15s each time, the extension was 100s at 72°C, and the denaturing annealing extension in the previous part was 15 cycles. The follow-up procedure is denaturation at 98°C for 15 s, annealing at 55°C for 15 s, extension at 72°C, and extension for 100 s, a total of 30 cycles, full extension for 5 min, and heat preservation at 12°C.

The PCR system was 5×PrimeSTAR GXL Buffer 10 μL, upstream primer F1 μL, downstream primer R1 μL, PrimeSTAR GXL DNA Polymerase 1 μL, template 0.1 μL, dNTP Mixture 4 μL, DMSO 2.5 μL, ddH ₂ O3 0.5 μL (each reagent was purchased from Takara); after the PCR program was completed, sample 1 μL for agar Sugar gel electrophoresis, the results are shown in Figure 1. Both the target gene and the pET-28a(+) vector obtained by direct recovery by PCR were double-digested with restriction endonucleases NdeI and XhoI, and the double-digested products were subjected to agarose gel electrophoresis, recovered by tapping the gel, and the recovered enzyme-digested products were ligated with T4 DNA ligase. The ligation product was introduced into competent cells DH5α for transformation, and 700uL of anti-antibiotic-free LB liquid medium was added to the transformed centrifuge tube. After gentle shaking at 37°C for 60 minutes, the bacterial solution was centrifuged at 5000rpm for 2 minutes, and 600uL of supernatant was discarded from each tube. Positive transformants were picked on a plate containing Kan resistance, transferred to 5 mL of liquid LB medium (containing Kan 50ug/mL) for overnight culture for 12 hours, and then the plasmid was extracted for double enzyme digestion verification. The verification picture is shown in Figure 2. The plasmid in Figure 2 was sent to Shanghai Jieli Biotechnology Co., Ltd. for sequencing. The nucleotide sequence of the target gene obtained is shown in SEQ ID NO.1, with a length of 1614bp and named SNA15.

Example 2

Expression and purification of recombinant protein SNA15 protein

The vector plasmid successfully constructed in Example 1 was transferred into the competent cell BL21, and the empty plasmid was introduced into the competent cell BL21 for empty control, and the template gene (SEQ ID NO.2 in CN110804089) was constructed by the same method and transferred into the competent cell BL21 as a positive control. The E. coli transformation procedure was the same. Centrifuge the two tubes of bacterial solution at 5000rpm for 2min, discard 600uL of supernatant in each tube, use the remaining culture medium to suspend and precipitate the bacterial cells, spread evenly on the screening plate containing Kan resistance, and place the two plates at 37°C for overnight culture. Then pick a single colony from the resistant plate and inoculate it in 5mL liquid medium (containing Kan 50ug/mL) for overnight culture. After 16 hours, transfer the Escherichia coli cultured in two tubes of 5mL liquid medium (containing Kan50ug/mL) to 800mL liquid LB medium (containing Kan 50ug/mL) for cultivation. The culture conditions are 37°C, 180rpm, and cultured for about 4h. When the OD reached 0.6, IPTG with a final concentration of 0.05mM was added for induction, and the induction conditions were 16°C, 180rpm for 16h. Under the conditions of 4°C and 1.5MPa, three cycles of crushing were carried out, and then a sonicator was used in an ice-water bath, with 400W, working for 2s, and an interval of 8s as a cycle of ultrasonication for 30min. Afterwards, centrifuge at 10000rpm, 50min, 4°C to get the supernatant, and take an equal amount of the solution for SDS-PAGE electrophoresis. The results are shown in Figure 3a, 1: supernatant of E. coli containing the recombinant vector; 2: supernatant of untransformed E. coli. It can be seen from Figure 3 that the size of the successfully expressed protein is 57.7 kDa, which is consistent with the expected size of the protein. The size of the protein (positive control) before modification in Figure 3b is 120kDa, and the band in Figure 3b is the band after nickel column purification. It can be seen that its expression level is low, and its property is found to be unstable after purification. The protein concentration of BBI protein quantification reagent (Shanghai Sangong) was used to measure the protein concentration. It was found that the expression level of SNA15 protein was high, which was more than 10 times the expression level before artificial modification, and it was found that the SNA15 protein protein was stable in the process of protein purification. The crushed supernatant of SNA15protein was subjected to nickel column affinity chromatography, and the target protein was hung on the column, washed and eluted. Before hanging the column, treat the column to ensure that there is no protein on the column, then balance it with the lysate, and then use the supernatant after centrifugation to pass through the nickel column, repeat 3 times, so that the target protein in the supernatant is hung on the column, and then wash the impurities with 20mM imidazole prepared from the cleavage buffer until the G-250 does not turn blue when the effluent is detected by G-250. So far, the target protein is eluted, and the target is preliminarily purified by nickel column affinity chromatography. The samples obtained by nickel column affinity chromatography will contain nucleic acids and more impurity proteins. In order to obtain more pure and better quality samples, the obtained samples need to be further purified by ion exchange chromatography. Ion exchange chromatography is used to concentrate the protein. Because the protein solution eluted by the nickel column is too large to be directly loaded on the ion exchange chromatography, it needs to be concentrated to concentrate the large volume to the volume required for loading. Before loading the sample, the protein purification instrument AKTA needs to be equilibrated for anion chromatography. Equal volumes of different diluted samples were taken for SDS-PAGE electrophoresis, and the results are shown in Figure 4. The results showed that the obtained protein sample had only one single band, indicating that the purified recombinant protein SNA15 protein had a relatively high purity and only had one single band, and its amino acid sequence was shown in SEQ ID NO.2.

Example 3

Synthesis, Characterization and Analysis of Silver Nanoparticles

Collect the target protein with only a single band obtained in Example 2, mix the collected pure components of the target protein, add 14 mg of silver nitrate to the 5 mL synthesis system, and then add the SNA15 protein solution with a final concentration of 152 ug/mL, and add 152 ug/mL of purified original protein solution (template protein before transformation, that is, the positive control of Example 2) to the other control tube, and the remaining solution is made up to 5 mL with ultrapure water, and the pH is adjusted to 12, the temperature is 30 ° C, and the rotation speed is 250 r pm, reaction time 16h can synthesize nano-silver, do not add target protein in the blank control group, other conditions are consistent with the experimental group, measure its characteristic absorption peak and the color of the synthesized nano-silver solution, A, B, C shown in Figure 5 are respectively the nano-silver synthesized by SNA15 protein, the nano-silver synthesized by the original protein (positive control), and the control group does not add any protein. It can be seen from Fig. Plasmon resonance is caused by the synergistic vibration of the electron cloud on the surface of the particle, and there is usually an absorption peak within 410-440nm. In general, the smaller the particle, the more the position of the absorption peak shifts forward, which indicates that the particle size of the nano-silver synthesized by the modified protein is smaller and the synthesis effect is better, and it can be seen from Figure 5b that the color of the nano-silver synthesized by the modified protein is higher in color (A is dark brown, B is light yellow, and C is transparent). Adjust different reaction temperatures (20-60°C) according to the above method, as shown in Figure 5c, the temperatures of A, B, C, D, and E are 30°C, 20°C, 40°C, 50°C, and 60°C, respectively. In the nano-silver synthesis experiment carried out under the same pH, it can be seen from the image that the synthesis condition at 30°C is the best. In addition, the protein solution eluted by anion chromatography in Example 2 was further removed from the influence of Nacl on the nano-silver synthesis experiment, and Nacl was replaced by gradient dialysis by dialysis, and the replaced protein solution was controlled at 152ug/mL, and nano-silver was synthesized under the same conditions as above to obtain a nano-silver solution.

The nano-silver synthesized by SNA15 protein is characterized by SEM as shown in Figure 6. Figure 6 shows that the overall dispersion effect of the synthesized nano-silver in the solution is good, and there is no large-scale agglomeration phenomenon. After the sample is ultrasonically subjected to TEM transmission electron microscopy, TEM is shown in Figure 7. It shows that nano-silver was successfully synthesized, and the particle size distribution is 2-25nm.

Example 4

Application of synthetic nano-silver in antibacterial aspect

Cultivate Staphylococcus aureus and Escherichia coli to the logarithmic phase. When the two bottles of solid LB medium are about to solidify, add Escherichia coli and Staphylococcus aureus with a volume of 1% of the medium volume, shake well, pour them into a disposable plate, wait for the plate to solidify and place the Oxford cup on the solid LB plate. When the Saccharomyces cerevisiae cultured to the logarithmic phase is about to solidify, add 1% of the medium volume Saccharomyces cerevisiae and shake well, then pour it into a disposable plate, wait for the plate to solidify and place the Oxford cup on the solid YPD plate made. The Penicillium spores cultured on the PDA slant of the eggplant bottle for 7 days were scraped off with 30mL sterile water, diluted twice, and 200uL of the diluted spore liquid was taken and spread on the PDA plate. All plates took the same volume of 100 μL of the nano-silver solution prepared in Example 3 and added it to the Oxford cup. 1-AgNPs was the nano-silver solution obtained by directly carrying out the nano-silver synthesis experiment of anion chromatography eluted protein, and 2-AgNPs was the anion chromatography eluted protein dialyzed to remove Na cl The nano-silver solution synthesized by interfering with the synthesis experiment, ampicillin sodium was added at a working concentration of 100ug/mL to 100 μL, and the SNA15 protein protein solution was added to 100 μL. The LB plates were cultured at 37°C, the YPD plates were cultured at 30°C, and the PDA plates were cultured in a constant temperature and humidity incubator at 28°C; the culture time of Escherichia coli and Staphylococcus aureus was 12 hours, the culture time of Saccharomyces cerevisiae was 24 hours, and the culture time of Penicillium was 72 hours. Inhibitory ability, and the antibacterial effect of nano-silver obtained by removing Nacl is stronger.

Example 5

The synthesis process of Example 3 was adopted, with the difference that the substrate concentration of silver nitrate was 14 mM, the reaction temperature range for synthesizing nano-silver was 20° C., the rotation speed was 100 rpm, the pH was 9, and the reaction time was 24 hours.

Example 6

Using the synthesis process of Example 3, the difference is that silver nitrate is used as the substrate with a concentration of 30 mM, the reaction temperature range for synthesizing nano-silver is 50° C., the rotation speed is 250 rpm, the pH is 13, and the reaction time is 16 hours.

Under the above conditions, nano-silver can be successfully synthesized, and each synthesis condition is very stable.

In summary, the shape of the silver nanometer prepared by the present invention is spherical nanosilver, wherein the synthetic nanosilver reaction is a non-enzyme-catalyzed nanosilver synthesis reaction, the particle size range of the nanosilver is 2-25nm, and the synthetic particle size range is controllable, and the surface of the synthesized nanosilver is coated with protein. This shows that the nano-silver has good biological activity, and according to domestic and foreign literature reports, nano-silver can be used to resist tumors, has excellent anti-tumor effect, and is expected to be used in the treatment of tumors, such as the inhibition of lung cancer cells and breast cancer cells. However, this biologically active nanosilver has very low toxicity to normal macrophages, and it is expected to develop new anticancer agents. At the same time, nanosilver has excellent antibacterial properties and has a broad spectrum, and has broad prospects in many fields such as food preservation and aquaculture.

sequence listing

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Val Lys Asp Asn Thr Lys Leu Glu Asn Thr Met Leu Asp Tyr Lys Glu

85 90 95

Val Ile Glu Asn Ala Asp Arg Val Ala Pro Thr Met Asp Ser Lys Ser

100 105 110

Trp Asn Thr Ser Gln Arg Arg Val Val Ile Lys Phe Ser Glu Lys Met

115 120 125

Asp Val Glu Thr Leu Ser Asn Tyr Ser Asn Tyr Leu Val Asp Ile Asn

130 135 140

Gly Gln Leu Arg Ser Glu Leu Ala Thr Lys Asn Arg Lys Leu Asn Asn

145 150 155 160

Val His Ile Leu Gly Ser Asp Asn Ser Asp Phe Val Glu Ile Ala Thr

165 170 175

Lys Thr Asn Asp Val Asn Ile Glu Val Glu Asn Ala Phe Asp Ile Gly

180 185 190

Gly Ser Ser Asn Thr Leu His Thr Val Leu Ala Leu Asn Asp Gly Ser

195 200 205

Lys Leu Gly Glu Ser Thr Thr Gly Phe Glu Tyr Lys Ser Lys Val Ala

210 215 220

Ala Glu Thr Ile Asn Ile Ile Thr Phe Ala Gln Lys Ile Lys Gly Gly

225 230 235 240

Ser Tyr Ser Ala Ala Leu Val Asp Arg Lys Thr Val Glu Ile Lys Phe

245 250 255

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

260 265 270

Gly Ser Thr Asn Thr Ile Ser Ser Ile Glu Ala Asn Gly Thr Ser Ser Thr

275 280 285

Val Lys Val Lys Phe Asp Lys Glu Ile Asn Ala Asp Ala Ser Asn Phe

290 295 300

Glu Leu Asn Val Lys Phe Asp Lys Leu Val Thr Leu Ala Gly Asp Ser

305 310 315 320

Phe Thr Gly Ser Asp Val Ile Lys Ala Pro Thr Lys Thr Val Ala Gly

325 330 335

Asn Leu Leu Asp Lys Val Ala Pro Glu Val Val Gly Asp Ala Asn Tyr

340 345 350

Ala Thr Asn Gln Ala Asp Lys Thr Ile Leu Val Pro Phe Thr Glu Glu

355 360 365

Leu Thr Leu Asn Gly Ser Ser Ala Glu Leu Ala Ala Asn Asp Phe Lys

370 375 380

Val Val Arg His Ser Asp Arg Lys Thr Leu Thr Ala Gly Ser Asp Tyr

385 390 395 400

Thr Val Lys Leu Ser Pro Asp Asn Lys Gly Val Leu Ile Thr Ile Lys

405 410 415

Ala Ala Asp Thr Thr Val Gly Val Leu Asp Val Asn Lys Ile Asp Ser

420 425 430

Ala Asn Ala Gln Leu Ser Tyr Ala Glu Val Tyr Ser Gly Ala Phe Ile

435 440 445

Ala Ala Glu Ser Thr Arg Glu Ser Ala Asn Ala Ile Asp Phe Gly Ala

450 455 460

Ala Thr Ala Lys Thr Ala Thr Val Ser Gly Ser Tyr Lys Thr Thr Gly

465 470 475 480

Pro Thr Asp Val Glu Ala Asn Glu Thr Leu Val Phe Thr Phe Ser Ser

485 490 495

Glu Asp Asp Ser Thr His Ile Ala Asn Leu Lys Thr Asp Phe Ala Thr

500 505 510

Val Phe Lys Pro Glu Thr Thr Lys Arg Ala Ala Asp Gly Phe Thr Val

515 520 525

Ala Leu Glu His His His His His His His His

530 535

<210> 3

<211> 109

<212>DNA

<213> Artificial Sequence

<400> 3

ggaattccat atggatcgaa ctcgcccaaa agataagaaa gtaaaagtga aaaattcaaa 60

aactttagtt gtgactttct ctaaaacatt agattcttca gatggaaac 109

<210> 4

<211> 108

<212>DNA

<213> Artificial Sequence

<400> 4

ccgctcgaga gctacagtaa atccgtcagc tgctctcttc gtagtttctg gtttgaatac 60

agttgcaaag tcagttttaa gattagcaat atgagttgaa tcatcttc 108

<210> 5

<211> 3252

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atggcaaagc aaaacaaagg ccgcaaattt tttgcagcaa gcgcaacagc tgcattagtt 60

gcatctgcaa tcgtacctgt agcatctgct gcacaattaa acgacttcaa caaaatctct 120

ggctacgcta aagaagcagt tcaatcttta gtagacgctg gtgtaatcca aggggatgct 180

aacggcaact tcaacccact taaaactatc tcacgtgcgg aagctgctac aatcttcact 240

aacgctctag aattagaagc agaaggtgat gtaaacttca aagacgttaa agctgatgct 300

tggtactacg atgctatcgc agcaactgta gaaaacggaa tttttgaagg tgtaagtgct 360

actgaattcg caccaaacaa acaattaact cgttctgaag ctgctaaaat tttagtagat 420

gctttcgaat tagagggtga aggcgatcta agcgaattcg ctgacgcttc tactgttaaa 480

ccatgggcta aatcttacct agaaatcgca gttgcaaacg gcgttatcaa aggttctgaa 540

gcaaatggta aaacaaactt aaacccaaat gctccaatta ctcgccaaga cttcgcagtt 600

gtattctcac gtactattga aaacgtagat gctactccaa aagttgacaa aatcgaagta 660

gttgacgcta aaactttaaa cgttacttta tctgacggta ctaaagaaac tgttacttta 720

gaaaaagctt tagagcctaa caaagaaaca gaagttactt tcaaaattaa ggatgttgaa 780

tacaaagcta aagttactta tgttgtaact acagctactg cagttaaatc tgtatctgca 840

actaacctta aagaagtagt agttgaattc gacggtaaag ttgataaaga aacagcaact 900

gataaagcaa actattcttt aaaatcaggt aaagtaatta aatctgttaa gcttttagac 960

gatgagaaaa cagttgttct tacattagaa gatagactaa acaacaataa agttgatgca 1020

gttagtgttt ctaatgtaaa agctggcact ttaactattt cagctaaaaa tgtagaattc 1080

aaagcagttg ataacgaaat tccaactgtt aaagaagtaa aatctttagg aacaaaagct 1140

cttaaagttg tattctcaga accagttgat gatgtaaaac aaggaaactt cactttagac 1200

ggtaaagctt tctatggtaa agttactgtt actggaaatg aggtagtatt aactccttac 1260

agcacatcag cattagctgt tggtactcat tcattacaag tttctcaaat taaagacttt 1320

gcaggattca cttctcttac atctacaact gaatttactg tagtggaaga taaagaagct 1380

ccaacagtaa ctgaatcaag tgcgactctt gaaacactta ctttaacatt ctcagaagat 1440

gttgatccag attctatttc agcttctaaa gtattctgga aatcaggttc tgataaaaaa 1500

gctgctaaat ctgtagagcg tgttgaaggt aataaatata aatctacttt tgaaggagta 1560

aacactcttc caactggatc tgtaacagtt tatgttgaag gagtaaaaga ttactctggt 1620

aatacaattg ctgcggatac aaaagtagtt gtttcaccac aagttgatca aactcgtcca 1680

gaagttaaga aagtataagt gaaaaattca acaactttag ttgtgacttt ctctaaaaca 1740

ttaggttctt cagctggaaa caaaaacaac tatactgttc ttgataaaga cggaaaagtt 1800

atttctgtta aagacgcagt attatctaac gataaaaaat ctgtaactgt aactctatat 1860

aaagagttat cagatggtaa gaacacatta gaagttaaga atgtaaaaga taacacaaaa 1920

ttagagaata caatgttaga ttataaagaa gttattgaaa atgctgacag agtagctcct 1980

acaatggatt caaaatcatg gaacacatct caaagacgag ttgtttattaa attctctgaa 2040

aaaatggatg ttgaaacatt atctaactac tctaattatc ttgtagatat taatggacaa 2100

ttacgttcag aattagctac taaaaatcgc aaattaaata atgttcatat tttaggttct 2160

gataattctg attttgttga aattgctacc aaaaccaatg atgttaatat tgaagttgaa 2220

aatgcttttg atattggtgg ttcttctaat actttacata ctgttttagc tttaaatgat 2280

ggttctaaat taggtgaatc tactactggt tttgaatata aatctaaagt tgctgctgaa 2340

actattaata ttattacttt tgctcaaaaa attaaaggtg gttcttactc tgctgcatta 2400

gttgatagaa aaactgttga aatcaaattt aatgctggca tcgaagatgt aaaagctggt 2460

ggtattactt tagctacagg tagcacaaat acaatttctt caattgaagc aaatggtact 2520

tcaactgtta aagtgaaatt tgataaagaa attaatgctg acgcttctaa tttcgaatta 2580

aatgtaaaat ttgataaatt agttacttta gctggtgatt catttacagg ttctgatgta 2640

atcaaagcgc caacaaaaac agtagctggt aatttattgg ataaagttgc tcctgaagtg 2700

gttggagacg ctaattatgc aacaaatcaa gctgataaaa caattttagt tccattatact 2760

gaagaactta cacttaatgg atcttctgca gaattagctg caaatgattt taaagttgtt 2820

cgtcacagtg atagaaaaac actaactgca ggttctgatt atactgtaaa actatctcct 2880

gataataaag gtgttttaat tactattaaa gcagcagata ctacagttgg tgttttagat 2940

gttaataaaa ttgattctgc taatgctcaa ttatcttatg ctgaagttta ttctggtgct 3000

tttattgctg ctgaatcaac tcgtgaatct gcaaatgcaa ttgacttcgg tgctgcaact 3060

gcaaaaactg ctacagttag tggatcatac aaaactactg gtcctacaga tgttgaagct 3120

aatgaaactt tagtatttac tttctcttct gaagttgatt caactcttat tgctgatctt 3180

aaaactgagt ttgcaactgt attcaaacca gaaactacta atggagctgc tgatggtttt 3240

acagttgctt ga 3252

Claims (10)

1. A gene for encoding synthesized nano silver protein is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

2. The gene for encoding synthetic nano silver protein according to claim 1, wherein the structure domain rearrangement of the protein is performed by spatially simulating the structure of known synthetic nano silver protein, analyzing the functions and actions of each structure domain, screening to obtain the structure domain capable of efficiently synthesizing nano silver, and the gene sequence for encoding rearranged protein after rearrangement is used as a template, and the gene for encoding the efficient synthetic nano silver protein with the sequence shown as SEQ ID NO.1 is obtained through multiple site-directed mutagenesis.

3. The gene for encoding synthetic nano-silver protein according to claim 2, wherein the site-directed mutagenesis primer is shown in SEQ ID NO. 3-4.

4. A protein encoded by the gene of claim 1 and capable of efficiently synthesizing bioactive nano silver, wherein the amino acid sequence of the protein is shown as SEQ ID NO.2.

5. A recombinant vector comprising the gene encoding a synthetic nanosilver protein of claim 1.

6. An engineering bacterium, which is characterized by comprising the gene for encoding the synthesized nano silver protein according to claim 1 or the synthesized nano silver protein according to claim 4 or the recombinant vector according to claim 5.

7. Use of the gene encoding the synthesized nano silver protein according to claim 1 in the preparation of nano silver by a biological method.

8. Use of the synthetic nano-silver protein of claim 4 for preparing nano-silver by biological method.

9. The use according to claim 7 or 8, wherein silver nitrate is used as a substrate, the substrate concentration is 14-30 mM, the reaction temperature range for synthesizing nano silver is 20-50 ℃, the rotation speed is 100-250 rpm, the pH is 9-13, and the reaction time is 16-24 h.

10. The use according to claim 7 or 8, wherein the nanosilver produced in the use is spherical nanosilver with a particle size distribution of 2-25nm, the surface of which is coated with proteins involved in the synthesis of nanosilver.