阅读说明：本技术 一种来源于球形赖氨酸芽孢杆菌的纳米银合成蛋白及其应用 (Nano-silver synthetic protein derived from spherical lysine bacillus and application thereof ) 是由 李相前 汪强 刘培 田霞 于 2019-10-18 设计创作，主要内容包括：本发明公开了一种来源于球形赖氨酸芽孢杆菌的纳米银合成蛋白,并公开了该纳米银合成蛋白的具体序列,本发明揭示了球形赖氨酸芽孢杆菌制备纳米银的合成机制,并成功扩增出其相应DNA,经过重组载体和原核表达,可以大量制备具有高效合成生物纳米银能力的细菌,具有极其广阔的应用前景和工业价值。(The invention discloses a nano-silver synthetic protein from spherical lysine bacillus and a specific sequence of the nano-silver synthetic protein, discloses a synthetic mechanism for preparing nano-silver by spherical lysine bacillus, successfully amplifies corresponding DNA of the nano-silver, can prepare a large amount of bacteria with the capability of efficiently synthesizing biological nano-silver by recombinant vectors and prokaryotic expression, and has extremely wide application prospect and industrial value.)

1. The nano-silver synthetic protein derived from the lysinibacillus sphaericus is characterized in that the amino acid sequence of the nano-silver synthetic protein is shown in SEQ ID No. 1.

2. The nano-silver synthetic protein derived from the lysinibacillus sphaericus is characterized in that the nucleotide sequence of the nano-silver synthetic protein is shown in SEQ ID No. 2.

3. A recombinant vector is characterized in that the vector comprises a nucleotide sequence shown as SEQ ID NO. 2.

4. A recombinant cell comprising a nucleotide sequence set forth in SEQ ID No. 2.

5. Use of the nanosilver synthesis protein derived from bacteria according to claim 1 or 2 in the preparation of nanosilver in a biological process.

6. The application of claim 5, wherein the synthesis reaction for preparing the nano silver is carried out at 35-45 ℃, the rotation speed is 150-200 rpm, the concentration of the substrate silver nitrate is 15-25 mM, the pH value is 9.0-12.0, and the reaction time is 24 h.

7. The use of claim 5, wherein the prepared nano silver is spherical in shape and has a particle size of 30-50 nm.

Technical Field

The invention belongs to the technical field of protein purification technology and genetic engineering, and particularly relates to a nano-silver synthetic protein derived from lysine bacillus sphaericus and application thereof.

Background

The nano material is a solid material consisting of nano particles, and after the nano material is formed in the concept of the 80 s, the nano material is greatly concerned, and the metal nano material is an important branch of the nano material and is represented by noble metals of gold, silver and copper, wherein the research result of nano silver is the most. The nano silver material is an important novel nano material, and the preparation method mainly comprises three methods, namely a physical method, a chemical method and a biological method. With the popularization of green and environmental protection concepts, biological methods for synthesizing nano silver particles by using biological systems are receiving attention. The stability and the dispersibility of the biological nano silver are greatly improved under the protection of biomolecules, and the biological nano silver has good biocompatibility, special physicochemical properties, excellent antibacterial and anti-inflammatory effects and lower biotoxicity, and has unique advantages and great significance in the field of biomedical materials. The research on the optimal synthesis conditions of the nano-silver particles and the deep understanding of the synthesis mechanism of the nano-silver have great significance for preparing more efficient and high-yield antibacterial silver nano-products.

The nano silver is used for cancer diagnosis: early diagnosis of cancer helps to control the development of cancer and the spread of tumor cells, and is crucial for the treatment of cancer. The nano silver has a Localized Surface Plasmon Resonance (LSPR), i.e., incident photons resonate with free electrons on the surface of the nano silver, and has strong light absorption and scattering ability in an ultraviolet-visible region, and an extinction spectrum of the nano silver is influenced by the properties of the nano silver, such as size, morphology, orientation and the like, and is also related to the dielectric environment in which the nano silver is located. The interaction of nanosilver with biomolecules can cause a small change in local refractive index, and this change can be very sensitively reflected in the LSPR extinction spectrum of nanosilver. This optical property enables nanosilver to be used as a biosensor to convert difficult-to-detect biological identification information into a measurable LSPR spectral peak shift signal. Compared with the traditional immunodetection method (such as enzyme-linked immunosorbent assay), the nano-silver LSPR biosensor has the remarkable advantages of simple and convenient operation, quick response and no need of biomarkers.

In addition, the nano silver has a surface-enhanced Raman scattering (SERS) effect, so that Raman signals of substances to be detected can be greatly enhanced, and trace detection and even single-molecule detection can be realized. JUN and the like design that the magnetic nano silver core-shell composite nano material can perform targeting, separation and SERS imaging on cancer cells. The BAMRUNGSAP and the like design fluorescence labeling aptamer coupling Au-Ag nanorods, and realize SERS and fluorescence detection and imaging of Hela cells.

Nano silver as angiogenesis inhibitor: angiogenesis plays a very important role in the development of many diseases, including cancer.

Antibacterial activity: silver causes bacterial cells to undergo a high degree of morphological and structural changes leading to cell death. The nano silver can be attached to the cell wall of bacteria and the cell membrane containing sulfur elements, once the nano silver is combined with the membrane structure, the semi-permeability of the membrane is changed due to the silver-sulfur interaction formed by the nano silver on the membrane, and the cell content flows out due to osmotic pressure. Part of the nano silver particles can enter the bacteria to act with phosphorus-containing structures such as DNA, RNA and the like, so that the DNA is concentrated and the bacteria reproduction is inhibited, and the nano silver particles in the solution can release trace silver ions to inhibit the activity of enzyme in the bacteria and the expression of ATP (adenosine triphosphate) production related protein.

Anti-cancer properties: the most promising of the nanotechnology is to improve the design standard of cancer imaging technology and therapeutic drugs, reveal the tissue, morphology and function of biological systems on the nanometer scale, and the combination of nano silver particles and targeted drugs with the embedding technology for treating cancer is an important research direction of the nanoparticles in the medical field in the future. Through the coating technology, the nano silver particles can present a standard functionalized surface, and various matrixes can be added on the surface. For example, the nano silver particles coated with silicon dioxide can be connected with the substrate through stable ether bonds and ester bonds, so that the substrate is prevented from being decomposed by naturally metabolized enzymes.

The preparation method of nano silver can be divided into two main categories according to the reaction mechanism: physical and chemical methods. The physical method mainly uses physical means such as mechanical grinding, radiation and the like to change the silver elementary substance into a nanometer size. Although the principles of various physical methods are simple, they are limited in their use due to the high requirements for instrumentation and equipment, and the high cost of production. The chemical method is widely applied to the preparation of nano silver, and mainly comprises a chemical reduction method, a physical reduction method and a biological reduction method. Chemical reduction is one of the more widely used methods in laboratories and industry today. Reducing silver from its compounds or salts with reducing agents, the silver salt used being mainly AgNO₃Or its complex, the reducing agent mainly includes sodium borohydride, hydrazine hydrate, formaldehyde, polyol, ascorbic acid and metals active than Ag, etc., the dispersing agent or protective agent polyvinylpyrrolidone (PVP), sorbitol, polyvinyl alcohol, etc. are added to control the particle size and shape of the product. Reagents used in chemical reduction may have harmful effects on the environment or organisms in the using process, so an environment-friendly and green silver nanoparticle preparation scheme is in urgent need to appear.

Disclosure of Invention

The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide a nano-silver synthetic protein from lysine bacillus sphaericus and obtain a nucleotide sequence for coding the protein.

The invention also aims to solve the technical problem of providing the application of the nano-silver synthetic protein derived from the lysine bacillus sphaericus in preparing nano-silver by a biological method.

The technical scheme is as follows: a nano-silver synthetic protein derived from bacteria has an amino acid sequence shown as SEQ ID No. 1.

A nano-silver synthetic protein derived from bacteria has a nucleotide sequence shown as SEQ ID No. 2.

A recombinant vector comprises a nucleotide sequence shown as SEQ ID NO. 2.

A recombinant cell, which comprises a nucleotide sequence shown as SEQ ID NO. 2.

The application of the nano-silver synthetic protein derived from bacteria in preparing nano-silver by a biological method.

Preferably, the synthesis reaction temperature for preparing the nano silver is 35-45 ℃, the rotation speed is 150-200 rpm, the concentration of the substrate silver nitrate is 15-25 mM, the pH value is 9.0-12.0, and the reaction time is 24 hours.

Preferably, the prepared nano silver is spherical, and the particle size is 30-50 nm.

Has the advantages that:

the invention discloses a protein capable of efficiently synthesizing biological nano-silver in bacillus sphaericus, which is named as hypo-synthetic protein (lysine bacillus sphaericus), and the amino acid sequence of the putative protein is shown as SEQIDNO: 1. In addition, since NCBI does not describe a nucleotide sequence corresponding to the protein, the invention designs a degenerate primer by taking the protein sequence as a template, extracts a Lysinibacillus sphaericus genome as a PCR reaction template, searches the PCR reaction condition, successfully amplifies DNA corresponding to the protein and carries out sequencing, thereby also disclosing a nucleic acid sequence corresponding to the hyposynthetic protein for the first time. The biological nano-silver solution obtained by the method has the advantages of light yellow color, stable property, uniform size and particle size distribution of 30-50 nm, and the synthesis efficiency of the biological protein (lysine bacillus sphaericus) to nano-silver is higher under the conditions of pH of 9.0-12.0 and temperature of 35-45 ℃, and the synthesis time is about 1 day. And extracellular proteins are attached, so that the biological function is good. The invention purifies and discovers key protein which plays a role in the reaction of synthesizing the nano-silver by the spherical lysine bacillus, discloses a synthesis mechanism of preparing the nano-silver by the bacteria, successfully amplifies corresponding DNA of the nano-silver, can prepare a large amount of bacteria with the capability of efficiently synthesizing the biological nano-silver by a recombinant vector and prokaryotic expression, and has extremely wide application prospect and industrial value.

Drawings

FIG. 1 is a color change (A) of dialyzed protein solution synthesized nano silver at different saturation degrees and a peak image (B) of dialyzed protein solution synthesized nano silver at each group of saturation degrees;

FIG. 2 is an SDS-PAGE protein electrophoresis of dialyzed protein solutions at 80% saturation;

FIG. 3 is a graph of the nanosilver synthesis peaks for each set of eluents and eluents;

FIG. 4 is an SDS-PAGE protein electrophoresis of a 200mM eluted protein solution;

FIG. 5 is agarose gel electrophoresis of the amplified gene of interest;

FIG. 6 is an SDS-PAGE protein electrophoresis of recombinant bacteria supernatants and a control;

FIG. 7 is a diagram of the synthesis peaks of nano-silver of the supernatant of the recombinant bacteria and the control group.

Detailed Description

The invention will be further described with reference to the following implementation steps and the accompanying drawings.

Materials, reagents, and the like used in the following embodiments are commercially available unless otherwise specified.