CN106755042B

CN106755042B - Preparation method of bioactive small peptide based on combined self-shearing and protein scaffold

Info

Publication number: CN106755042B
Application number: CN201611243905.7A
Authority: CN
Inventors: 马兴元; 李尚洁; 郑文云; 刘地; 陈潇潇; 李妍瑶
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2016-12-29
Filing date: 2016-12-29
Publication date: 2020-10-09
Anticipated expiration: 2036-12-29
Also published as: CN106755042A

Abstract

The invention discloses a preparation method of bioactive small peptide based on combined self-shearing and protein scaffold, which comprises the following steps: (1) constructing an ELPs-mediated C-intein fusion LfcinB recombinant plasmid; (2) preparing two kinds of fusion antibacterial peptides of Titin-LfcinB and Telethion-LfcinB by taking escherichia coli as a host, and mixing to prepare ZT-LfcinB. The invention couples expression, purification and stable utilization together, so that the whole small peptide is assembled into the medicament with the stable bracket from the construction of an expression vector to the in vitro, the invention is an integral process, and the practical problem that the expression production and utilization efficiency of certain proteins and small peptides is generally lower is better solved.

Description

Preparation method of bioactive small peptide based on combined self-shearing and protein scaffold

Technical Field

The invention belongs to the field of biological medicine and disease immunity, and particularly relates to a preparation method of bioactive small peptide based on combined self-shearing and protein scaffold.

Background

Since the discovery of penicillin in the 1940 s, the widespread use of antibiotics has freed people from the fear of bacterial infections. But with the long-term use and abuse of antibiotics, the generation of drug resistance of pathogenic microorganisms is stimulated. Drug-resistant tubercle bacillus, MRSA, etc. are increasingly harmful to human and animals, and cause disastrous economic loss to society. The progress in the selection of new antibiotics has also become increasingly difficult due to bacterial resistance.

In the case of slow development of synthetic antibiotics, how to overcome the problem of bacterial resistance caused by antibiotics is a hot spot of current research. Research shows that some organisms can quickly generate small molecular substances with antibacterial activity, namely antibacterial peptide, to participate in organism immunity after being infected by microorganisms. Scientists have subsequently identified thousands of antimicrobial peptides from plants, microorganisms, amphibians, insects, mammals, and even humans, suggesting that it is an important immune molecule for almost all living species. The antibacterial peptide has biological activities of broad-spectrum antibiosis, antivirus, anticancer cells and the like, and is completely different from the antibiotics generated by traditional microorganisms (bacteria, fungi, streptomyces and the like) in the aspects of amino acid composition, synthesis mechanism, action mechanism and the like. Therefore, antibacterial peptides, which are small molecular peptides synthesized by ribosomes and are of biological origin, have undoubted potential advantages in the field of development of novel antibacterial agents, and are becoming a research hotspot in the biomedical science [1 ]. People actively explore the medicinal value of the antibacterial peptide, hope to develop a novel antibacterial agent for replacing the traditional antibiotics, and provide a new way for solving the problem of the drug resistance of pathogenic bacteria to the antibiotics.

In 1972, a research group led by the swedish scientist Boman H.G. discovered and purified the antimicrobial peptides P9A and P9B in induced Bombyx mori immune hemolymph by injecting E.coli into Xigubia pupae. The primary structure of this antimicrobial peptide was determined by amino acid sequence sequencing analysis and was named cecropins (cecropins), the first antimicrobial peptide found and obtained in humans.

Antimicrobial peptides (AMPs) are composed of 10-50 amino acids, and are important components of a biological congenital nonspecific defense system. AMPs have the characteristics of broad-spectrum antibacterial activity, high efficiency, stability and the like, and have strong inhibition effect on bacteria, fungi, even HIV and cancer cells. In addition, since the target of action is mainly located on the cell membrane of the microorganism, the cell membrane depolarization of the microorganism is induced, and the cell rupture is caused, so that the generation of microorganism resistance mutation is not easy to cause, and the antibiotic is one of the strong candidates for replacing the traditional antibiotics.

At present, people mainly obtain target antibacterial peptide by three methods, namely (1) biological extraction, (2) chemical synthesis and (3) expression by using a genetic engineering means, wherein the methods have advantages and disadvantages.

(1) Biologically extracted antimicrobial peptides

The disadvantages of the induction production of antibacterial peptide in vivo are low content, high difficulty, low efficiency, high requirements for technology and cost, difficulty in realizing large-scale production, and unrealistic for the production of some higher organisms and humanized antibacterial peptides.

(2) Chemically synthesized antimicrobial peptides

With the further research on the chemical structures of polypeptides and proteins and the advent of polypeptide synthesis technology, the method for artificially synthesizing active peptides provides great convenience for the research on antibacterial peptides. Many studies have shown that chemically synthesized AMPs show the same biological activity as natural AMPs, but the chemical synthesis method also suffers from high cost, which limits the wide application of antimicrobial peptides.

(3) Genetically engineered antibacterial peptides

After analysis and comparison of a large number of antibacterial peptide molecular structures, people find that computer-aided design of novel antibacterial peptides and preparation of the novel antibacterial peptides by utilizing a genetic engineering technology are an important means for new drug development. The antibacterial peptide has the advantages of small molecular weight, simple chemical structure, wide antibacterial spectrum, no antibiotic resistance, low immunoreactivity and convenient synthesis, and is favorable for testing, analyzing, modifying and reconstructing an antibacterial mechanism by using computer software.

The rapid development of genetic engineering technology makes the large-scale preparation of antibacterial peptides possible, but many problems exist at present. The molecular weight of the antibacterial peptide expressed in bacteria by a gene engineering technology is small, and an expressed product is possibly harmful to a host, so that the high-level expression of genes is adversely affected. How to effectively reduce the toxicity of the antibacterial peptide expression product to host bacteria and improve the expression efficiency is a problem to be solved by utilizing the genetic engineering expression of the antibacterial peptide. In the fusion expression of host bacteria, the application of affinity chromatography is the most extensive. Conventional affinity chromatography is to fuse an affinity tag to the protein sequence of interest at the DNA level. Then the cell disruption solution passes through an affinity column to be separated to obtain the target protein. However, the affinity media used in chromatographic techniques are expensive and often require the removal of the affinity tag from the fusion protein by proteolytic hydrolysis. For industrial applications, the removal of affinity tags is the most expensive step in the protein production process. The addition of a foreign substance may also affect the biological activity of the protein of interest. In addition, the antibacterial peptide has small molecular weight and is easy to degrade, the recovery rate is further reduced, and the production cost of the polypeptide is greatly improved. Therefore, a novel antibacterial peptide expression and purification system is urgently needed to be developed, and a foundation is laid for industrial preparation of antibacterial peptides.

Elastin-like polypeptides (ELPs) are artificial biopolymers adapted from the gene level, which are derivatives of Elastin (elastins) widely present in vertebrate connective tissue. The elastin-like protein is composed of one repeating pentapeptide unit Val-Pro-Gly-Xaa-Gly, where Xaa may be any amino acid other than proline. The system obtained by fusing the N-terminus or C-terminus of the functional target protein with ELP is called ELPy.

(1) Structure and physicochemical Properties of Elastin-like proteins (ELPs)

ELPs are polymers composed of Val-Pro-Gly-Xaa-Gly (VPGXG) pentapeptide as a unit, and are derivatives of elastin. Where Xaa in turn calls the residue "guest residue", it can be an amino acid other than proline (Pro) because proline disrupts the phase transition. The literature also classifies ELPs based on the type and number of occurrences of the fourth amino acid. In order to make the structural labeling of a particular ELP appear concise and standard, the following expressions have been proposed: [ XiYjZk ] n

X, Y, Z-each letter represents the amino acid present at the fourth position of the pentamer;

i, j, k-represents the ratio of the fourth amino acid present in the pentameric unit;

n-represents the total number of ELP pentamers given.

ELPs belong to one of three classes of thermosensitive biopolymers, whose properties change with changes in ambient temperature. Water-soluble ELPs have a lower critical solution temperature, i.e., when the temperature is higher than the so-called phase transition temperature, the ELPs change from a soluble state to a coagulated state within a narrow temperature range (2-3 ℃), a process called coacervation. Meyer defines the phase transition temperature Tt as: the temperature value at which the solution turbidity increased to half of the initial value as a result of increasing the solution temperature to cause the ELP to coagulate is the phase transition temperature. When the solution temperature is below Tt, the free polymer chains are in a disordered hydrated state (soluble form); when the solution temperature is higher than Tt, the polymer chains take on a more ordered structure (beta-helix) and become stabilized by hydrophobic interactions, while the intramolecular beta-structure also strengthens the bonding ability between the polymer chains.

However, the above process is reversible, and the ELP also changes from the condensed state to the soluble form when the ambient temperature is lowered. Interestingly, this temperature-dependent reversible phase transition property still exists even when ELP is fused to other proteins. The G and L proteins were fused to the ELP domain and found to be reversible in phase transition over a range of temperatures. The Tt value of ELPs depends on several factors. Such as the concentration of ELP in the solution, the ionic strength of the solution, the salt concentration and the molecular mass of the polymer, and furthermore the properties of the fusion protein have a certain influence on the Tt value. As these parameter values increase, Tt decreases. For a given biopolymer, the difference in the fourth amino acid of the monomer also has an effect on the Tt value. When the amino acid at this position belongs to a hydrophobic group, Tt is lower; conversely, when the group is hydrophilic, the Tt value increases (the effect depends on the molar ratio of the particular residue). By varying the fourth residue of the pentapeptide unit, the desired value of Tt can be designed. If the amino acid residue at the fourth position is susceptible to ionization, the Tt value can be adjusted by changing the pH; while other molecules can be coupled to ELP when the amino acid has a side chain that can undergo a chemical reaction.

(2) Intein sources and structures and functions

In 1990, the groups of Hirata and Kane, respectively, coded for the vacuolar membrane H of Saccharomyces cerevisiae⁺An in-frame insert was found in the VMA1 gene of ATPase and was present in the precursor mRNA, translated with the Vmal protein, and excised after translation. This process of self-excision is called splicing of the protein, since it is similar to the process by which the RNA intron is cleaved from the precursor mRNA. The cleaved-off segment of the polypeptide is designated intein. Thus, inteinsIs a protein element embedded within a host protein, having the ability to self-splice (self-splicing), also called a protein splicing element. When the self-splicing reaction occurs, the intein is cut from the host protein by itself, and the two sides of the extein are connected into a complete protein through side chains and the function of the host gene is restored. Cleavage of inteins is a post-translational process that does not require the assistance of enzymes or cofactors. Self-splicing of proteins involves only four intramolecular reactions in which only a small proportion of the key catalytic residues of inteins and exteins are involved. Since 12 months 1999, over 100 inteins have been identified and included in the InBase database (http:// www.neb.com/neb/inteins. html).

Inteins can be structurally divided into two classes: 1) macromolecular inteins containing homing endonucleases in two splice regions; 2) missing the mini-intein of the homing endonuclease. Recently, an intein that can undergo trans-splicing has been newly discovered. A homing endonuclease is a double-stranded DNA endonuclease present at a specific site which promotes lateral migration between the genomes of its own coding regions through flanking sequences in a recombination-dependent process called "homing". Typically, the homing endonuclease is encoded by an open reading frame comprising introns or inteins. The macromolecular inteins are bifunctional proteins with a protein cleavage region and a central endonuclease region. Chong et al deleted the central endonuclease region in the macromolecular intein, constructed the mini-intein with efficient splicing function, and demonstrated that the endonuclease region did not participate in the splicing of proteins. The splicing region is divided by the endonuclease region into two subdomains, N-terminal and C-terminal, with a conserved region of amino acids A, N2, B and N4 in the N-terminal domain and G and F in the C-terminal domain. These regions are also present in the mini-intein. The natural mini-intein and the artificially constructed mini-intein are similar in three-dimensional structure. Known inteins are less similar in sequence and are only highly conserved at the N-and C-terminal sequences. Most intein residues begin with Ser or Cys and end with His-Asn or His-Gln.

Intein splicing is a rapid four-step affinity attack mediated by four conserved splicing functional residues: (1) the first residue at the N-terminus of the intein, N-O (serine, Ser) or N-S acyl (cysteine, Cys), is transferred to form a (thio) ester linkage between the N-extein and the intein domain. (2) (thio) ester bonds are attacked by the OH-or SH-group of the first residue of the C-extein (Cys, Ser or Thr) to transesterify the side chain of the first residue of the N-extein with that of the C-extein. (3) The C-terminal conserved amino acid residue Asn of the intein generates cyclization to release the intein, and the two sides of the extein are connected by (sulfur) ester bonds. (4) (thio) ester bonds linking the two exopeptides are rearranged and the S-N or O-N acyl group is converted to form a peptide bond.

(3) Self-cleavage of inteins and uses

The natural intein can generate self splicing (splicing) in vivo, and the mutation of the conserved amino acid sequence at the N-terminal or C-terminal of the natural intein by using a genetic engineering means can control the self-cutting reaction only at the C-terminal or N-terminal. If only N-terminal cleavage is desired, the terminal residue of the native intein is mutated to avoid step (3) of the splicing machinery (while step (4) is also terminated). The first two steps can still occur, so that ester bonds are spontaneously hydrolyzed to cut the N-terminal exopeptide from the intein. Conversely, by mutating the first residue of the intein, neither of steps (1) (2) or (4) of the splicing mechanism occurs, but step (3) is continued, thereby cleaving the extein from the C-terminus. By utilizing the characteristics, the genetic engineering intein with self-cutting ability can be fused with various protein purification tags to mediate the purification effect of the protein.

Engineered inteins can be divided into two classes: pH-induced C-terminal self-cleaving inteins and thiol-induced N-/C-terminal self-cleaving inteins. pH-induced inteins are the most economical system because the cleavage reaction can be induced by adjusting the pH of the buffer. Among them, SspDNaB inteins carried by pTWIN1 and pTWIN2 plasmids developed by New England Biolabs (NEB) are currently the most published pH-induced self-cleaving inteins. Another pH-induced intein is the engineered, genetically screened mutant mini-intein Δ I-CM from Mut RecA by Branki et al, which has been successfully combined with non-affinity chromatography tags such as ELP, PHB, etc. for protein purification. The drawback of pH and temperature induced inteins is that the proteins may self-cleave in vivo before being recovered and purified. However, the extent of cleavage in vivo can be limited to a minimum by low temperature expression (12-15 ℃).

The most reported thiol-induced inteins are the Sce VMA inteins from saccharomyces cerevisiae, which are commercially available and can be used as IMPACT Kit in combination with CBD affinity tags. The cleavage reaction at the N-terminus can be induced by strong nucleophiles such as 2-mercaptoethane, sodium sulfonate, hydroxylamine, thiophenol, beta-mercaptoethanol, DTT or free cysteine, with DTT being the most commonly used. The advantage of using thiol-induced inteins is that there is little self-cleavage reaction in vivo, and in addition, pH and temperature induced inteins are not thiol sensitive, inexpensive, and suitable for large-scale production. The disadvantage is that thiol induction may destroy the disulfide bonds of the protein of interest.

The intein has wide application value in the field of biotechnology. The natural splicing activity of inteins, which is known as intein-mediated protein ligation (IPL) or Expressed Protein Ligation (EPL), is well established for the application of molecular biology and biotechnological methodologies in the ligation of proteins and polypeptides. In addition, inteins are also widely used for segmentation labeling of proteins, protein cyclization, controlled expression of toxic proteins, conjugation of proteins to quantum dots, bioseparation, and the like in NMR analysis. In basic studies, researchers use inteins to monitor protein-protein interactions in vivo, or to alter the location of proteins into organelles. Most inteins in biotechnological applications are derived from prokaryotic microorganisms, or are engineered variants of the Saccharomyces cerevisiae VMA 1-intein.

Thomas et al applied methanobacterium mini-intein (Mth RIR1, intein) for protein ligation. The Mini-intein contains 134 amino acid residues. Having a proline Pro adjacent to the N-terminal cysteine of the intein^-1-Cys¹In the native state, it was found that its splicing efficiency in E.coli is low. When proline is mutated to alanine, the splicing (spicing) efficiency of the intein increases. And mutation of arginine at C-terminal or cysteine at N-terminal of intein into alanineWhen in acid, the mutant shows a high-efficiency cleavage effect (cleavage) at the N-or C-terminal. Mutation Asn of the C-terminal/N-terminal of mini-intein¹³⁴-Ala/Cys¹Ala, and is fused with CBD, the other end is fused with MBP or T4ligase to form the triplets of MBP-intein (N) -CBD and CBD-intein (C) -T4DNA ligase, and the triplets are expressed by using escherichia coli, the proteins are extracted by using CBD after the cells are crushed, and finally the two triplets are mixed under a certain condition, and the MBP and the T4ligase are connected through the self-polypeptide bond reaction of the two triplets. Experiments show that the mini-intein has the self-splicing ability, and the unique property expands the application of an intein-mediated method in the connection of in vitro bacteria expression macromolecular fusion proteins.

Wu et al expressed and purified disulfide-bond-containing human antibody fragments, maltose-binding protein and β -galactosidase using pH-induced Δ I-CM intein. They fused the target protein from the gene level to a self-cleaving intein tag with a chitin binding domain and purified by chitin-agarose affinity column chromatography. The purified target protein remains on the column, and the cleaved target protein can be directly eluted by changing pH8.5 to pH6.0-6.5 at room temperature. Experiments prove that the CBD-intein purification method can obtain good yield and product purity, and the product activity is good.

(4) Titin/Z1Z2-Telethonin compound protein scaffold and application

Titin, also called as Titin, is the largest protein encoded by a single gene discovered at present, has the molecular weight of about 3-3.7MDa and the length of about 1 mu m, occupies about half of the sarcomere, is a highly elastic molecule and plays an important role in maintaining the integrity of the muscle sarcomere. Titin is ubiquitous in cardiac and skeletal muscle, is called tertiary myofilament, has complex biomechanical properties and biochemical functions, and is a hotspot of molecular biology and clinical medicine research in recent years. Titin consists of 300 repetitive domains in tandem, mainly consisting of immunoglobulin (Ig) and fibronectin type III domains, PEVK (proline, glutamic acid, valine and lysine rich) flexible domain and several other distinct domains. It was found that deletion of the C-terminus of Titin, including the kinase domain, damages myofibrils and leads to the development of muscle diseases such as muscle weakness.

Telothonin, also known as TCAP, is encoded in human by the TCAP gene and is expressed in the Z-line of cardiac and skeletal muscle and functions to regulate sarcomere assembly, T-tubule function and apoptosis. Telethonin contains 167 amino acid residues and has a molecular weight of 19 kDa. It has been found to be associated with the development of several diseases, including myodystrophy, hypertrophic cardiomyopathy, dilated cardiomyopathy and idiopathic cardiomyopathy. Telethonin has a unique beta-sheet structure that allows it to be in an antiparallel relationship with the Z1Z2 domain of cardiac and skeletal muscle Titin Titin.

It was found that the N-terminus of Titin is composed of two immunoglobulin domains Z1 and Z2, while Z1Z2 binds tightly to Teleth onin. Chemical bond studies show that the latter binds with high specificity to the Titin complex Z1Z2, but does not interact with the monomers of both. Biochemical studies of Telethonin have shown that the binding of Telethonin to the Z1Z2 complex is a prerequisite for the stimulation of muscle growth. The Morten Bertz et al study found that the cleavage force between Titin and Telethonin was as high as 700pN, far exceeding all other Ig regions measured from Titin. The Titin-telethonin complex perfectly anchors the megamyoprotein firmly to the Z-line. Zhouqijian et al, by means of X-ray crystallography, revealed how the amino acid residues of the giant myoprotein Titin assemble with Telethonin into an antiparallel 2:1 sandwich structure. The unique structure of the wrinkle was confirmed by in vivo protein interaction assay, and in vitro fluorescence resonance energy transfer (see FIG. 1). Moreover, inspired by the structure, the wrinkle-resistant hydrogel also designs a platform which takes a Titin/Z1Z2-Teleth protein complex as a drug carrier to research and develop a protein drug, and is called ZT technology. The research on Titin/Z1Z 2-Teletsonin shows that the size of the compound is about 56KDa and the compound can be prepared by prokaryotic host expression. The source is human source, the immunogenicity is low, the binding force between molecules is firm, and the protein is super stable; as a carrier, the carrier can greatly improve the half-life and stability of protein and polypeptide drugs, and is a potential carrier for developing protein drugs.

The invention is inspired by the previous basic research, on the basis of preparing a series of active protein polypeptides such as antibacterial peptide and the like by a genetic engineering means, a special fusion protein label, namely intein (intein) with a self-cutting function, Elastin (ELPs) with conditional phase transition and ZT complex (Titin/Z1Z 2-Telethin) with multiple connection points, is combined and optimized, and is applied to preparing bioactive proteins such as antibacterial peptide and the like as a whole, so that the toxicity problem of directly expressing the antibacterial peptide to a host is avoided; the traditional affinity chromatography purification method is avoided, the cost is saved, and the advantages of strong binding force, high solubility and stability and the like of the bracket are utilized, so that conditions are created for preparing active protein polypeptides such as antibacterial peptide and the like on a large scale in an industrialized mode. Particularly, the technology has certain universality, and can be widely applied to efficient preparation and stable utilization of various bioactive protein polypeptides, thereby greatly overcoming the bottleneck problem commonly existing in polypeptide preparation and utilization.

Disclosure of Invention

The first purpose of the invention is to provide a preparation method of bioactive small peptide based on combination of self-shearing and protein scaffold.

The second purpose of the invention is to provide the application of the bioactive small peptide prepared by the preparation method of the bioactive small peptide based on the combined self-shearing and protein scaffold.

The third purpose of the invention is to provide the bioactive small peptide prepared by the preparation method of the bioactive small peptide based on the combination of self-shearing and protein scaffold.

In order to achieve the above purpose, the invention discloses the following technical scheme: a method for preparing bioactive small peptide based on combined self-shearing and protein scaffold is characterized by comprising the following steps:

(1) construction of ELPs-mediated C-intein fusion LfcinB recombinant plasmid: constructing a cow milk ferritin high-efficiency fusion expression system taking escherichia coli as a host, inserting a pET-22b-EI vector into LfcinB constructed by overlapping PCR to construct pET-22b-EI-LfcinB, and obtaining the LfcinB without exogenous amino acid residues under the self-shearing action of C-Inteins mediated protein by utilizing the reversible phase transition characteristic of ELPs;

(2) according to the characteristics of the Titin protein and the Telethion protein, the recombinant plasmid is respectively connected with LfcinB in series to construct pET-24a-Titin-LfcinB and pET-24 a-Telethion-LfcinB recombinant plasmids, and two fusion antibacterial peptides of Titin-LfcinB and Telethion-LfcinB are prepared by taking escherichia coli as a host, wherein Titin-LfcinB: mixing the Telethion-LfcinB monomers according to the molar weight ratio of 1-4:1-4 to prepare ZT-LfcinB, wherein 1-4 monomers are stably coupled together in series or in parallel, and Native SDS-PAGE analysis shows that the assembly is successful.

As a preferred embodiment, tini-LfcinB: the molar weight ratio of Telothonin-LfcinB is 2: 1.

As a preferred embodiment, EI-LfcinB is optimally expressed under the condition that the target protein is optimally expressed when IPTG is induced at a concentration of 1mM, an induction temperature of 25 ℃ and an induction expression time of 24 hours.

As a preferred embodiment, the cleavage buffer used for self-cleavage of inteins is a pre-cooled NaCl (1-3M) -containing cleavage buffer.

In order to realize the second purpose of the invention, the invention discloses the following technical scheme: the bioactive small peptide prepared by the method for preparing the bioactive small peptide based on the combined self-shearing and protein scaffold is applied to preparing antibacterial or antiviral drugs.

In order to achieve the third purpose of the invention, the invention discloses the following technical scheme: the bioactive small peptide is prepared by a bioactive small peptide preparation method based on combined self-shearing and protein scaffold.

The invention is divided into the following three parts:

(1) design and construction of expression vector containing self-shearing functional intein, phase-changeable elastin-like protein and protein scaffold polypeptide Titin/Z1Z2-Teleth nin

Constructing a bovine milk ferritin high-efficiency fusion expression system taking escherichia coli as a host, and respectively inserting LfcinB constructed by overlapping PCR and two multi-copy tandem sub-fragments of 2LfcinB and 4LfcinB synthesized by gene into the pET-22b-EI vector existing in a laboratory to successfully construct three expression plasmids of pET-22b-EI-LfcinB, pET-22b-EI-2LfcinB and pET-22b-EI-4 LfcinB. The three forms of fusion antibacterial peptides are expressed, and LfcinB without exogenous amino acid residues is obtained under the self-shearing action of C-Inteins mediated protein by utilizing the reversible phase change characteristic of ELPs. According to the characteristics of the Titin protein and the Telethion protein, the recombinant plasmid is respectively connected with LfcinB in series to construct pET-24a-Titin-LfcinB and pET-24 a-Telethion-LfcinB recombinant plasmids, two kinds of fusion antibacterial peptides of Titin-LfcinB and Telethion-LfcinB are prepared by taking escherichia coli as a host, and the ZT-LfcinB fusion antibacterial peptide is prepared by assembling according to a certain proportion.

(2) Purification-tag-free expression, temperature-controlled phase change separation and purification and activity detection of active small molecule polypeptide

The invention utilizes the temperature-dependent phase transition cycle (ITC) characteristic of ELPs, can be used as a protein purification tool to replace the traditional affinity chromatography technology, and saves the process cost. The C-Inteins intein with temperature and pH dependence can mediate the self-shearing action of the target protein to separate the target protein, thereby avoiding the interference caused by introducing protease or other reagents and simplifying the protein separation step and the cost. The Intein polypeptide fusion expression mode mediated by ELPs ensures that the antibacterial peptide is in an inactive state in cells, thereby avoiding the inhibition effect of the antibacterial peptide on host cells; the expression product is used for measuring the activity of the recombinant antibacterial peptide by an agar plate bacteriostasis ring method and MIC. And the steps are simple and convenient, the time and the purification cost are saved, and the method is a hotspot for industrial scale amplification.

(3) Titin and Telethonin are respectively connected with target polypeptide in series to improve the stability utilization of the antibacterial peptide. Titin-LfcinB and Telethion-LfcinB genes are constructed by overlapping PCR, and two expression plasmids of pET-24a-Titin-LfcinB and pET-24 a-Telethion-LfcinB are successfully constructed. After the target protein is expressed, the Titin-LfcinB is expressed by the supernatant, the total is 244aa, the molecular weight is about 26KDa, and the fusion polypeptide can be separated and purified by using a nickel column affinity chromatography method. After exploring the protein purification conditions, the optimal imidazole elution concentration is 400mM, the purity is high, and the yield is 1.27mg/100ml of zymocyte liquid. Telechonin-LfcinB is expressed by inclusion bodies, has 156aa in total and has the molecular weight of about 17.8 KDa. Washing with inclusion body washing buffer solution for three times to obtain the fusion polypeptide with high purity, and after urea gradient dialysis renaturation, ultrafiltering and concentrating, wherein the yield is 907.9 mug/100 ml fermentation liquor. According to the molar mass of Titin-LfcinB: Telethonin-LfcinB is prepared by mixing the two monomers at a ratio of 2:1, and Native SDS-PAGE analysis shows successful assembly. Because the Telethion-LfcinB is very easy to form precipitates in the assembly process, a small amount of Titin-LfcinB fusion protein monomer is remained in the supernatant, which lays a foundation for expressing the antibacterial peptide with stable activity by using a prokaryotic host.

The invention has the advantages that: the invention provides a preparation and utilization technology of bioactive small peptide combined with protein scaffold by self-shearing. On the basis of preparing the antibacterial peptide by a genetic engineering means, the invention constructs a stable and efficient fusion expression system of bioactive small peptide by taking escherichia coli as a host by utilizing the self-shearing characteristic of Elastin-like polypeptides (ELPs) mediated endopeptides, applies a protein scaffold ZT technology to prokaryotic expression of the antibacterial peptide, and creates conditions for the stability, multiple effects and efficient utilization of the bioactive small peptide expressed by ZT fusion. The expression, purification and stable utilization are coupled together, so that the whole small peptide is assembled into the medicament with the stable bracket from the construction of an expression vector to the in vitro, the whole process is an integral process, and the practical problem that the expression production and utilization efficiency of certain proteins and small peptides is generally low is better solved. The technology provides a new way and selection for overcoming the defects of low expression efficiency, poor stability, toxicity to a host, poor stability, small molecular weight, short utilized half-life and the like of the bioactive small peptide represented by the antibacterial peptide in the aspect of genetic engineering research. Meanwhile, the invention has certain universality and practicability, the application field of the bioactive small peptide prepared by utilizing the combined technology is not limited to infectious diseases such as antibiosis, antivirus and the like, and the application object is not limited to human beings and can also be used for animals.

Drawings

FIG. 1 is a schematic diagram of Titin/Z1Z 2-Telethin complex.

FIG. 2 is a map of pET-22b-EI-LfcinB recombinant plasmid.

FIG. 3 shows colony PCR of ET-22b-EI-2LfcinB recombinant transformant, M:500DL Marker; 1-6: PCR of single-colony bacteria liquid; 7: and (5) negative control.

FIG. 4 shows PCR verification of recombinant transformant colonies of pET-22b-EI-4LfcinB, M:500DL Marker; 1-9: PCR of single-colony bacteria liquid; 10: and (5) negative control.

FIG. 5 is a schematic diagram of pET-22b-EI-4LfcinB recombinant plasmid construction.

FIG. 6 shows 12% SDS-PAGE of EI-LfcinB protein purified by ITC technique, M: protein marker; 1: precipitating after cell disruption; 2: first ITC post-supernatant; 3: adding a precooling buffer and then centrifuging and precipitating; 4, precipitating EI-LfcinB after the second ITC.

FIG. 7 is a recombinant LfcinB protein Tricine-SDS-PAGE, M: protein marker; 1: recombinant LfcinB.

FIG. 8 shows IPTG induced concentration optimized SDS-PAGE, M low molecular weight protein marker; 1: 0.2mM IPTG; 2: 0.4mM IPTG; 3: 0.6mM IPTG; 4: 0.8mM IPTG; 5: 1.0mM IPTG.

FIG. 9 shows IPTG induction temperature optimized SDS-PAGE, M low molecular weight marker; 1: 37 ℃; 2: 30 ℃; 3: 25 ℃; 4: 20 ℃; 5: 16 ℃ is adopted.

FIG. 10 is an IPTG induction time optimized SDS-PAGE, M: low molecular weight protein marker; 1: inducing for 6 h; 2: inducing for 18 h; 3: inducing for 24 hours; 4: inducing for 32 h; 5: induction was carried out for 48 h.

FIG. 11 shows 12% SDS-PAGE of EI-2/EI-4 LfcinB protein purified by ITC technique, M: protein marker; 1/1': cell lysate prior to induction; 2/2': post-induction cell lysate; 3/3': first ITC precipitation.

Fig. 12 is a bacterial growth curve assay, a. staphylococcus aureus growth curve, after addition of recombinant LfcinB; B. e.coli growth curves.

Fig. 13 is the bacteriostatic activity detection of recombinant LfcinB, a. 1: the concentration of recombinant LfcinB is 112 mug/ml; 2: the concentration of recombinant LfcinB is 56 mug/ml; 3: the concentration of the recombinant LfcinB is 28 mug/ml; 4: the concentration of recombinant LfcinB is 14 mug/ml; 5: positive control AMP 10. mu.g/ml. Anti-escherichia coli inhibition zone of LfcinB. 1: the concentration of recombinant LfcinB is 112 mug/ml; 2: the concentration of recombinant LfcinB is 56 mug/ml; 3: the concentration of the recombinant LfcinB is 28 mug/ml; 4: the concentration of recombinant LfcinB is 14 mug/ml; 5: positive control AMP 10. mu.g/ml.

Fig. 14 MIC assay of recombinant LfcinB, MIC of a.lfcinb against staphylococcus aureus; MIC of LfcinB against E.coli.

FIG. 15 PCR amplification of Titin-LfcinB gene, M: DL2000DNA marker; 1, performing first PCR on Titin-LfcinB; 2, second PCR of Titin-LfcinB.

FIG. 16 shows a map of pET-24a-Titin-LfcinB recombinant plasmid.

FIG. 17 PCR amplification of Teletsonin-LfcinB gene, M: DL2000DNA maker; 1, Telethonin first PCR; telethonin second PCR.

FIG. 18 shows the PCR amplification result of transformant bacterial liquid, M:2000bp Marker; 1-7 monoclonal transformants.

FIG. 19 is a map of a recombinant plasmid pET-24 a-Telethion-LfcinB.

FIG. 20Bradford method for determination of protein concentration standard curves.

FIG. 21Titin-LfcinB protein 15% SDS-PAGE, M: protein maker; 1: before induction; 2: guiding the bacteria liquid; 3: centrifuging the supernatant of the crushing liquid; 4: centrifuging and precipitating; 5: flowing a nickel column through the liquid; 6: 400mM imidazole flow through; 7: 400mM imidazole eluent; 8: concentrating the solution after ultrafiltration.

FIG. 22 Teletsonin-LfcinB protein 15% SDS-PAGE, M: protein maker; 1: inducing to obtain a bacterial liquid; 2: centrifuging the supernatant at 10000 rpm; 3: a first inclusion body wash; 4: a second inclusion body wash; 5: the inclusion bodies are denatured and dissolved in solution.

FIG. 23ZT-LfcinB protein 15% Native SDS-PAGE, M: protein maker; 1: ZT-LfcinB is combined for 48h at 4 ℃; 2: Telethonin-LfcinB; 3: and (3) concentrating Titin-LfcinB by ultrafiltration.

FIG. 24 is a graph showing the bacteriostatic activity test of ZT-LfcinB, and the bacteriostatic zone of ZT-LfcinB against Escherichia coli. 1: the experimental group ZT-LfcinB is 500 mu g/ml; 2: positive control AMP 5. mu.g/ml; 3: positive control AMP 10. mu.g/ml ZT-LfcinB anti-Staphylococcus aureus zone. 1-3: the experimental group ZT-LfcinB is 500 mu g/ml; 4: negative control group ddH 2O; 5: positive control AMP 5. mu.g/ml.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The quantitative tests in the following examples are all set up in three or more repeated experiments, and the results are averaged.

Example 1 construction of ELPs-mediated C-intein fusion LfcinB recombinant plasmids

Original mCherry genes are cut off by using pET-22 b-EI-mChery plasmids existing in laboratories through restriction endonucleases, and then genes of LfcinB, 2LfcinB and 4LfcinB obtained by PCR amplification or whole gene synthesis are replaced, so that three recombinant expression plasmids of pET-22b-EI-LfcinB, ET-22b-EI-2LfcinB and pET-22b-EI-4LfcinB are constructed. The results of nucleic acid electrophoresis verification and sequencing show that the recombinant plasmid is successfully constructed.

3LfcinB gene sequence, because the length of LfcinB is only 75bp, 25 amino acid residues and the amino acid sequence is FKCRRWQW RMKKLGAPSITCVRRAF (SEQ ID NO: 1), two longer primers can be directly designed, and the target gene can be obtained by utilizing overlapping PCR. The primer design is as follows:

LfcinB-F：

CAGCGTGTACACAACATGTTCAAATGCCGTCGTTGGCAGTGGCG

BsrGI(SEQ ID NO：2)

LfcinB-R：

CCCAAGCTTTTACCATGGAAAGGCACGACGCACACAGGTGATAGACG

HindIII(SEQ ID NO：3)

2LfcinB amino acid sequence and gene sequence

Amino acid sequence: FKCRRWQWRMKKLGAPSITCVRRAF-NNNNN (linker) FKCRRWQWRMKKLGAPSITCVRRAF (SEQ ID NO: 4)

The gene sequence is as follows:

TGTACACAACTTCAAATGCCGTCGTTGGCAGTGGCGTATGAAAAAACTGGGTGCTCCGTCTATCACCTGTGTGCGTCGTGCCTTTAACAACAACAACAATTTCAAATGCCGTCGTTGGCAGTGGCGTATGAAAAAACTGGGTGCTCCGTCTATCACCTGTGTGCGTCGTGCCTTTTAAAAGCTT(SEQ ID NO：5)

4LfcinB amino acid sequence and gene sequence: FKCRRWQWRMKKLGAPSITCVRRAF-NNNNN (linker) -FKCRRWQWRMKKLGAPSITCVRRAF-NNNNN (linker) -FKCRRWQWRMKKLGAPSITCVRRAF-NNNNN (linker) -FKCRRWQWRMKKLGAPSITCVRRAF (SEQ ID NO: 6)

The gene sequence is as follows:

TGTACACAACTTCAAATGCCGTCGTTGGCAGTGGCGTATGAAAAAACTGGGTGCTCCGTCTATCACCTGTGTGCGTCGTGCCTTTAACAACAACAACAATTTCAAATGCCGTCGTTGGCAGTGGCGTATGAAAAAACTGGGTGCTCCGTCTATCACCTGTGTGCGTCGTGCCTTTAACAACAACAACAATTTCAAATGCCGTCGTTGGCAGTGGCGTATGAAAAAACTGGGTGCTCCGTCTATCACCTGTGTGCGTCGTGCCTTTAACAACAACAACAATTTCAAATGCCGTCGTTGGCAGTGGCGTATGAAAAAACTGGGTGCTCCGTCTATCACCTGTGTGCGTCGTGCCTTT TAAAAGCTT(SEQ IDNO：7)

TABLE 1 primers

Construction of pET-22b-EI-LfcinB recombinant plasmid

LfcinB overlap PCR amplification: two long primers of LfcinB-F and LfcinB-R are designed, the C-end has a 20bp overlapping sequence, and the two primers are combined through the overlapping part by annealing to obtain the full length of the LfcinB gene through extension. Electrophoresis results show that a strip appears at 80-100bp, which is consistent with expected results, and the amplification is successful.

Construction of pET-22b-EI-2LfcinB recombinant plasmid: the plasmid pUC57-2LfcinB containing 2LfcinB synthesized by whole gene is transferred into escherichia coli DH5 alpha for competence and culture, and a monoclonal transformant is picked up for amplification to extract the plasmid. The double digestion with the corresponding restriction enzymes BsrGI and HindIII, 3% agarose gel electrophoresis to obtain the single band of the target gene of 2LfcinB, subsequent agarose gel recovery kit to recover the target gene fragment, and the overnight connection with the linearized plasmid pET-22 b-EI. The ligation product was transformed into competent DH 5. alpha. and after plating, single clones were picked for PCR in bacterial suspension followed by 3% agarose gel electrophoresis. The results are shown in FIG. 3. Sequencing by picking 1-2 bacterial liquid and feeding Ruidi biotechnology company shows that the target gene 2LfcinB is correctly connected to the vector, and the pET-22b-EI-2LfcinB recombinant plasmid is successfully constructed.

Construction of pET-22b-EI-4LfcinB recombinant plasmid: plasmid pUC57-4LfcinB containing 4LfcinB synthesized by the whole gene was transformed into E.coli DH 5. alpha. and cultured. The single clone transformant is selected for amplification, plasmids are extracted, and the restriction enzymes BsrGI and HindIII are used for double digestion. A single band of the target gene of 4LfcinB was obtained by 2% agarose gel electrophoresis, and the target gene was recovered by an agarose gel recovery kit and ligated with the linearized plasmid pET-22b-EI overnight. The ligation product was transformed into competent cells DH 5. alpha. and after plating, single clones were picked for PCR in bacterial suspension followed by 2% agarose gel electrophoresis. The results are shown in FIG. 4.

The sequencing of the 5-9 bacterial liquid by Ruidi biotechnology shows that the target gene 4LfcinB is correctly connected to the vector, and the pET-22b-EI-4LfcinB recombinant plasmid is successfully constructed. FIG. 5 is a spectrum of successfully constructed recombinant plasmids.

Example 2 expression and purification of EI-LfcinB

Expression of EI-LfcinB: after searching the expression conditions of the target protein, only EI-LfcinB was efficiently expressed in the three recombinant proteins EI-LfcinB, EI-2LfcinB and EI-4LfcinB, but neither EI-2LfcinB nor EI-4LfcinB could be expressed when introduced into two different expression hosts. Therefore, only the expression conditions of EI-LfcinB were investigated and LfcinB antibacterial peptide was successfully purified. The expression and purification results of EI-LfcinB are shown in FIG. 6.

Fig. 6 shows that EI-LfcinB is expressed in e.coli BL21(DE3), and the fusion protein EI-LfcinB can be obtained by two ITC cycles, with a size of about 67 KD. However, this method has some disadvantages, and it can be seen from the figure that a small amount of hetero-protein still exists in the fusion protein after purification by ITC.

Purification of recombinant LfcinB: the obtained fusion protein EI-LfcinB is put in a self-shearing buffer solution for 16h in a water bath at 22 ℃, the supernatant is collected, and the Tricine-SDS-PAGE result shows that the recombinant LfcinB antibacterial peptide with the purity of more than 95 percent is obtained. The concentration of the recombinant LfcinB is 2mg/100ml fermentation liquor measured by a biuret method.

Although a relatively pure fusion protein was not obtained after two ITC using EI tags, a band of impurities was still present under the target protein, but as can be seen from FIG. 7, after the Intein self-cleavage reaction was performed, the band of impurities was completely removed by ITC. The target polypeptide LfcinB with small molecular weight can be left in the supernatant. Thus, the recombinant antibacterial peptide with high purity can be obtained.

Example 4 optimization and purification of EI-LfcinB inducible expression conditions

In order to increase the EI-LfcinB production, we searched and optimized the expression conditions of pET-22b-EI-LfcinB/E.coli BL21(DE3), and the results are shown below.

(1) EI-LfcinB adding IPTG concentration optimization

Five experimental groups are designed, cultured for 2-3h at 37 ℃ and 200rpm in a constant temperature shaking table until OD is reached₆₀₀After reaching about 0.5, IPTG was added to each group of tubes at final concentrations of 0.2, 0.4, 0.6, 0.8, 1.0mM, respectively, and the mixture was transferred to 20 ℃ for induction expression for 24 hours. As a result of SDS-PAGE by directly collecting the cells, FIG. 8 shows that the highest expression level of EI-LfcinB was observed when the concentration of IPTG induced was 1.0 mM.

(2) EI-LfcinB adding IPTG induced expression temperature optimization

Five experimental groups are designed, cultured for 2-3h at 37 ℃ and 200rpm in a constant temperature shaking table until OD is reached₆₀₀After reaching about 0.5, IPTG was added to a final concentration of 1.0mM, and then 5 tubes were induced at 37 ℃ 30 ℃, 25 ℃, 20 ℃ and 16 ℃ for 24 hours. As a result of SDS-PAGE of the directly collected cells, FIG. 9 shows that the expression level of EI-LfcinB was the highest at 25 ℃.

(3) EI-LfcinB expression time optimization

Five experimental groups are designed and shaken at constant temperatureCulturing at 37 deg.C and 200rpm for 2-3h until OD₆₀₀After reaching about 0.5, IPTG was added to a final concentration of 1.0mM, and then 5 tubes were placed at 25 ℃ for induction expression for 6h, 18h, 24h, 32h, and 48 h. As a result of SDS-PAGE of the directly collected cells, FIG. 10 shows that the expression level of EI-LfcinB was the highest at 24 hours.

In conclusion, when the conditions for expression of EI-LfcinB were optimized, the target protein could be optimally expressed when the IPTG induction concentration was 1mM, the induction temperature was 25 ℃ and the induction expression time was 24 hours.

(4) Purification of EI-2LfcinB and EI-4LfcinB expression

As is clear from the results of SDS-PAGE in FIG. 11, although the recombinant plasmids of EI-2LfcinB and EI-4LfcinB were successfully constructed, the fusion proteins of the correct size could not be expressed. After 16h of self-shearing reaction, Tricine-SDS-PAGE was performed on the centrifuged supernatant, and no polypeptide electrophoretic band was obtained.

Example 5 detection of the bacteriostatic Activity of recombinant LfcinB

(1) Staphylococcus aureus and Escherichia coli growth curve assay

As can be seen from FIG. 12, the growth of the cells of the experimental group added with 64. mu.g/ml recombinant LfcinB was slower than that of the control group, indicating that the recombinant LfcinB prepared by the experiment has a certain bacteriostatic activity on gram-positive bacteria and gram-negative bacteria. And the bacteriostatic activity of LfcinB on gram-positive bacteria is stronger than that on gram-negative bacteria.

(2) Determination of LfcinB bacteriostatic activity by agar diffusion method

The bacteriostatic activity of the prepared recombinant LfcinB antibacterial peptide is determined by taking staphylococcus aureus and escherichia coli as indicator bacteria, and three groups of repeated experiments are carried out.

FIG. 13a shows the result of the agar diffusion method to determine the bacteriostatic activity of LfcinB against Staphylococcus aureus. FIG. 13b shows the results of the agar diffusion method for determining the bacteriostatic activity of LfcinB on Escherichia coli. The marked 1-4 respectively represent the inhibition zones of the oxford cups after 200ml of LfcinB with the final concentration of 112 mug/ml, 56 mug/ml, 28 mug/ml and 14 mug/ml is added for 18 hours; 5 shows the addition of 200ml of an AMP positive control at a final concentration of 10. mu.g/ml. The detection result of the agar diffusion method shows that the recombinant LfcinB antibacterial peptide has the bactericidal effect on both staphylococcus aureus and escherichia coli. The experimental results show that 112 mu g/ml of LfcinB can averagely generate a bacteriostatic circle with the diameter of 14mm for staphylococcus aureus and about 10mm for escherichia coli. With the reduction of the usage amount of LfcinB, the diameter of the inhibition zone is gradually reduced.

(3) Microdilution assay for Minimum Inhibitory Concentration (MIC)

The concentration of the purified recombinant LfcinB antibacterial peptide is 2mg/ml, and the recombinant LfcinB antibacterial peptide is subjected to two-fold serial dilution by using a sterile MH culture medium, mixed culture with an equal volume of a culture solution of staphylococcus aureus or escherichia coli, and MIC determination. Experimental results FIG. 14 shows that MICs of recombinant LfcinB were 56. mu.g/ml and 112. mu.g/ml for Staphylococcus aureus and Escherichia coli, respectively.

TABLE 2 MIC of recombinant LfcinB

Example 6 construction of ZT-LfcinB fusion antimicrobial peptides in E.coli

(1) Primer design

TABLE 3 Titin primers

TABLE 4 Telethonin primers

(2) Overlapping PCR amplification of Z-LfcinB

TABLE 5Z-LfcinB PCR reaction System (F2-R2)

TABLE 6 reaction conditions of Z-LfcinB (F2-R2)

The product was recovered using a PCR purification kit.

TABLE 7Z-LfcinB PCR reaction System (F1-R1)

TABLE 8 reaction conditions for Z-LfcinB (F1-R1)

(3) Overlapping PCR amplification of T-LfcinB

TABLE 9T-LfcinB PCR reaction System (F2-R2)

TABLE 10T-LfcinB reaction conditions (F2-R2)

The product was recovered using a PCR purification kit.

TABLE 11T-LfcinB PCR reaction System (F1-R1)

TABLE 12T-LfcinB reaction conditions (F1-R1)

(4) Recovery of target fragment and double enzyme digestion identification of expression vector

Linearization of pET-24a-Titin/Telethonin plasmid

TABLE 13 double restriction enzyme System

Reacting in water bath at 37 ℃ for 1h, adding 10 XDNA loading buffer to terminate the reaction, and adding a proper amount of Gelred.

(5) Target gene Titin-LfcinB double enzyme digestion

TABLE 14 double enzyme digestion System

(6) Double enzyme digestion of target gene Teletsonin-LfcinB

TABLE 15 double enzyme digestion System

(7) Ligation of the fragment of interest and the expression vector

And respectively carrying out agarose gel electrophoresis on the target fragment and the vector, carrying out gel recovery, and connecting the enzyme digestion products.

TABLE 16 ligation of the cleavage products

(8) Construction of pET-24a-Titin-LfcinB recombinant plasmid

Titin-LfcinB overlap PCR amplification, and the LfcinB gene is spliced to the 3' -end of Titin through two times of PCR. FIG. 15 shows that the electrophoresis results show that a band appears at 500-750bp, which is consistent with the expected result, and the amplification is successful.

(9) Construction of pET-24a-Titin-LfcinB recombinant plasmid

And (3) connecting the Titin-LfcinB target fragment obtained by PCR purification and recovery with a linearized plasmid pET-24a overnight, transforming competence DH5 alpha, performing plate-laying culture, selecting a monoclonal antibody for PCR verification of bacterial liquid, and performing 3% agarose gel electrophoresis. The colony PCR results showed that a band appeared around 1000bp, consistent with the expected length. Sequencing by selecting bacterial liquid No. 1, No. 3 and No. 4 from SenRuidi biotechnology company shows that the target gene tin-LfcinB is correctly connected to the vector, and the pET-24 a-tin-LfcinB recombinant plasmid is successfully constructed. FIG. 16 is a diagram of a successfully constructed recombinant plasmid.

(10) Construction of pET-24 a-Telethion-LfcinB recombinant plasmid

Telethonin-LfcinB overlap PCR amplification, and the LfcinB gene is spliced to the 3' -end of Telethonin by two times of PCR. Electrophoresis results show that a band appears at 500bp, which is consistent with the expected result, and the amplification is successful. The results are shown in FIG. 17.

And (3) constructing a pET-24 a-Telethion-LfcinB recombinant plasmid.

The Telethion-LfcinB target fragment obtained by the PCR purification and recovery is connected with a linearized plasmid pET-24a overnight, transformed into competent DH5 alpha, after the plating culture, a single clone is picked for the PCR verification of a bacterial solution, and then the 3% agarose gel electrophoresis is carried out. The results are shown in FIG. 18.

The colony PCR results showed that a band appeared around 750bp, consistent with the expected length. Sequencing by selecting 3, 4 and 5 bacteria liquid SenRuidi biotechnology shows that the target gene Telethion-LfcinB is correctly connected to the vector, and the pET-24 a-Telethion-LfcinB recombinant plasmid is successfully constructed.

Example 7 ZT-LfcinB fusion antimicrobial peptide expression, purification and Activity assay

(1) Expression of the protein of interest

picking BL21 transformant single colony to inoculate Kan-containing transformant single colony⁺(50. mu.g/ml) in 5ml of LB medium, at 37 ℃ and 200rpm, overnight;

② adding the overnight-cultured bacterial liquid into the culture medium containing Kan according to the proportion of 1:100⁺(50. mu.g/ml) in 100ml of LB medium Erlenmeyer flask, 37 ℃ at 200rpmShake culturing for about 2.5h to OD₆₀₀Reaching about 0.5;

③ adding IPTG to the final concentration of 1mM, inducing for 6h at 37 ℃ and 200 rpm;

fourthly, collecting the culture solution, centrifuging for 10min at 4 ℃ and 12000rpm, and removing the supernatant to collect thalli;

adding 15ml PBS to resuspend the thalli, centrifuging at 4 ℃ and 12000rpm for 10min, abandoning the supernatant, collecting the thalli, and preserving at 4 ℃ for later use;

sixthly, 10ml of precooled PBS is added to resuspend the thalli, and the cells are homogenized and broken under high pressure at 4 ℃.

(2) Purification of proteins of interest

1. Ni-NTA purification of Titin-LfcinB expressed in supernatant

Firstly, centrifuging the crushed bacterial liquid at 4 ℃ and 12,000rpm for 15min, and collecting the supernatant;

② assembling Ni columns, using ddH of 5 × column volume₂Washing 20% ethanol with O, and removing air completely;

③ using PBS buffer solution with 10 multiplied column volume to carry out column balance;

fourthly, slowly adding the protein sample, and filtering the supernatant with a filter membrane of 0.22 mu m before use;

slowly eluting with NTA gradient of 5

times column volume

20, 60, 100, 200, 300, 400, 600Buffer, collecting gradient flow-through liquid, and analyzing optimum elution condition by SDS-PAGE;

sixthly, eluting the column by NTA-600 with the volume of 10 multiplied by the column volume;

using ddH of 10 × column volume₂O, cleaning the column;

adding 20% ethanol solution, and storing at 4 deg.C;

ninthly, dialyzing and ultrafiltering and concentrating the purified and eluted protein, and storing at-20 ℃.

2. Purification of Inclusion-expressed Telethion-LfcinB

Firstly, centrifuging the crushed bacterial liquid at 4 ℃ and 10,000rpm for 15min, and collecting precipitates;

adding inclusion body washing liquid to resuspend and precipitate, stirring for 10min at 200rpm in ice bath, centrifuging for 15min at 4 ℃ and 8000rpm, and discarding supernatant;

thirdly, repeating for three times;

adding 10ml of inclusion body denaturation buffer solution, stirring for 30min by a 200rpm rotor under ice bath, centrifuging for 15min at 4 ℃ and 8000rpm, and taking supernatant;

fifthly, the supernatant is concentrated by ultrafiltration and stored at the temperature of minus 20 ℃;

sixthly, reserving samples in all steps and performing SDS-PAGE.

Determination of the protein concentration after purification by the Bradford method

Drawing of BSA Standard Curve

① 0, 0.2, 0.4, 0.6, 0.8 and 1.0ml Bovine Serum Albumin (BSA) standard solution (100 mu g/ml) into a test tube respectively, and ① and supplementing ddH₂O to 1 ml;

② adding 5ml Coomassie brilliant blue dye solution into each test tube, mixing uniformly, standing for 5-10min at room temperature;

measuring the light absorption value at 595nm by using a spectrophotometer, and recording the reading; the light absorption of the sample without BSA was used as a blank; a standard curve is plotted and the concentration of protein in the sample is calculated. The standard curve is shown in fig. 20.

TABLE 17 Standard Curve Table

(3) ZT-LfcinB self-assembly

And (3) determining the concentration of the purified and concentrated fusion antibacterial peptide according to the following formula: Telethonin-LfcinB was mixed at a ratio of 2:1 and self-assembled for 48h under dialysis. And concentrating the assembled ZT-LfcinB by ultrafiltration.

(4) ZT-LfcinB fusion antibacterial peptide activity determination

1. Agar diffusion method for determining ZT-LfcinB bacteriostatic activity

Adding agar powder into distilled water according to the proportion of 0.8%, sterilizing at the high temperature of 121 ℃, spreading about 7ml of agar powder into a sterile flat plate, and solidifying for later use;

② selecting single colony to be detected by inoculating loop, inoculating into 5ml MH broth culture medium, making OD at 37 deg.C and 200rpm for 3-6h₆₀₀＝0.6；

③ taking 0.5ml of the bacterial liquid to 5 percent according to the proportionthe bacterial count was maintained at 2-7 × 10 in 0ml of LB medium containing 1% agar⁵CFU/ml. Uniformly spreading the mixture on a culture dish by 15ml under aseptic condition, and completely solidifying for later use;

putting Oxford cups on the fully solidified agar plates, filtering the purified recombinant ZT-LfcinB by using a 0.22 mu m filter membrane, adding 200 mu l of the purified recombinant ZT-LfcinB into the corresponding Oxford cups, standing and culturing for 16-24h at 37 ℃, and determining the size of a bacteriostatic zone₂O was used as a negative control.

The experiment is repeated three times.

(5) Purification of ZT-LfcinB fusion antibacterial peptide

Purification of Titin-LfcinB: the Titin-LfcinB fusion antibacterial peptide is expressed by a supernatant, and the N-end of the Titin-LfcinB fusion antibacterial peptide is provided with a 6 XHis histidine tag, so that the Titin-LfcinB fusion antibacterial peptide is separated and purified by a nickel column affinity chromatography method. After the bacteria are broken, the supernatant is obtained by centrifugation, the supernatant is filtered by a 0.22 mu m filter membrane, the filtrate is slowly added into a nickel column and is eluted by imidazole gradient, and the SDS-PAGE analysis of the result in a figure 21 shows that the elution condition is optimal when the concentration of the imidazole is 400mM, and the purity can reach more than 95 percent. After concentration by centrifugation through an ultrafiltration tube, the protein concentration was measured by Brad-ford as 1.27mg/100ml of the fermentation broth.

(6) Purification of Teleth sonin-LfcinB

The Telethion-LfcinB fusion antibacterial peptide is expressed by an inclusion body, and is separated and purified by adopting an inclusion body washing method. After the bacteria are crushed, centrifuging to take the precipitate, adding an inclusion body washing buffer solution for washing three times, adding a denaturation buffer solution containing 8M urea to fully dissolve the fusion antibacterial peptide, and dialyzing by urea gradient for 24 hours to slowly renature. After ultrafiltration and centrifugal concentration, SDS-PAGE analysis shows that after three times of washing with inclusion body washing buffer solution, membrane protein and other impurities can be completely removed, and the purity can reach more than 95%. The protein concentration measured by Brad-ford was 907.9. mu.g/100 ml of the fermentation broth.

(7) ZT-LfcinB fusion antibacterial peptide

FIGS. 22 and 23 show that Titin-LfcinB and Telethin-LfcinB fusion polypeptides after respective ultrafiltration concentration are mixed according to the molar ratio of 2:1, the mixture is stood at 4 ℃ for assembly for 48 hours, supernatant is obtained after centrifugation at 12,000rpm, and native SDS-PAGE shows that ZT-LfcinB fusion polypeptides are formed. We found that small amounts of Titin-LfcinB fusion protein monomer remained in the supernatant as Teletsonin-LfcinB precipitated very easily during assembly. After concentration by centrifugation in an ultrafiltration tube, the protein concentration was measured by Brad-ford at 2 mg/ml.

(8) Determination of ZT-LfcinB fusion antibacterial peptide activity by agar diffusion method

The MIC of the prepared ZT-LfcinB fusion antibacterial peptide is determined by taking staphylococcus aureus and escherichia coli as indicator bacteria, and three groups of repeated experiments are carried out. FIG. 24a shows the result of the agar diffusion method for determining the bacteriostatic activity of ZT-LfcinB on Escherichia coli. The marked 1 in the figure represents the inhibition zone of the oxford cup after 200ml of ZT-LfcinB with the concentration of 500 mu g/ml is added for 18 h; 2 and 3 represent inhibition zones produced by adding 200ml AMP at a concentration of 5. mu.g/ml and 10. mu.g/ml to the Oxford cup, respectively. FIG. 24b is the result of the agar diffusion method for determining the bacteriostatic activity of ZT-LfcinB on Staphylococcus aureus. The marked 1-3 represents the inhibition zone after 200ml of ZT-LfcinB with the concentration of 500 mu g/ml is added into the oxford cup and acts for 18 hours; 4 represents 200ml of ddH₂O negative control, 5 shows the zone of inhibition by 200ml of 5. mu.g/ml AMP added. As can be seen from FIG. 24, there was still no macroscopic inhibitory effect on both test bacteria when the concentration of ZT-LfcinB was as high as 500. mu.g/ml.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

SEQUENCE LISTING

<110> university of east China's college of science

<120> preparation method of bioactive small peptide based on combined self-shearing and protein scaffold

<130>/

<160>17

<170>PatentIn version 3.5

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Phe Lys Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala Pro

1 5 10 15

Ser Ile Thr Cys Val Arg Arg Ala Phe

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cagcgtgtac acaacatgtt caaatgccgt cgttggcagt ggcgtatgaa aaaactgggt 60

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<213> Artificial Synthesis

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cccaagcttt taccatggaa aggcacgacg cacacaggtg atagacggag cacccagttt 60

tttcata 67

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<213> Artificial Synthesis

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Phe Lys Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala Pro

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Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala Pro Ser Ile

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Thr Cys Val Arg Arg Ala Phe

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<210>5

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tgtacacaac ttcaaatgcc gtcgttggca gtggcgtatg aaaaaactgg gtgctccgtc 60

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gctt 184

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<213> Artificial Synthesis

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Phe Lys Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala Pro

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Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala Pro Ser Ile

35 40 45

Thr Cys Val Arg Arg Ala Phe Asn Asn Asn Asn Asn Phe Lys Cys Arg

50 55 60

Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala Pro Ser Ile Thr Cys

65 70 75 80

Val Arg Arg Ala Phe Asn Asn Asn Asn Asn Phe Lys Cys Arg Arg Trp

85 90 95

Gln Trp Arg Met Lys Lys Leu Gly Ala Pro Ser Ile Thr Cys Val Arg

100 105 110

Arg Ala Phe

115

<210>7

<211>364

<212>DNA

<213> Artificial Synthesis

<400>7

tgtacacaac ttcaaatgcc gtcgttggca gtggcgtatg aaaaaactgg gtgctccgtc 60

tatcacctgt gtgcgtcgtg cctttaacaa caacaacaat ttcaaatgcc gtcgttggca 120

gtggcgtatg aaaaaactgg gtgctccgtc tatcacctgt gtgcgtcgtg cctttaacaa 180

caacaacaat ttcaaatgcc gtcgttggca gtggcgtatg aaaaaactgg gtgctccgtc 240

tatcacctgt gtgcgtcgtg cctttaacaa caacaacaat ttcaaatgcc gtcgttggca 300

gtggcgtatg aaaaaactgg gtgctccgtc tatcacctgt gtgcgtcgtg ccttttaaaa 360

gctt 364

<210>8

<211>19

<212>DNA

<213> Artificial Synthesis

<400>8

taatacactc actataggg 19

<210>9

<211>20

<212>DNA

<213> Artificial Synthesis

<400>9

tgctagttat tgctcagcgg 20

<210>10

<211>31

<212>DNA

<213> Artificial Synthesis

<400>10

cagcgtgtac acaacatgtt caaatgccgt c 31

<210>11

<211>38

<212>DNA

<213> Artificial Synthesis

<400>11

ggaattccat atgcatcatc atcaccacca tagcagca 38

<210>12

<211>49

<212>DNA

<213> Artificial Synthesis

<400>12

catatgcatc atcatcacca ccatagcagc atgaccaccc aggcaccga 49

<210>13

<211>89

<212>DNA

<213> Artificial Synthesis

<400>13

ccgctcgagt taaaaggcac gacgcacaca ggtgatagac ggagcaccca gttttttcat 60

acgccactgc caacgacggc atttgaaag 89

<210>14

<211>85

<212>DNA

<213> Artificial Synthesis

<400>14

tgccaacgac ggcatttgaa agaaccacca ccaccagaac caccaccacc agaaccacca 60

ccaccgccct gcaccagcag ctcgg 85

<210>15

<211>38

<212>DNA

<213> Artificial Synthesis

<400>15

ggaattccat atgaaacatc accatcacca tcacccca 38

<210>16

<211>25

<212>DNA

<213> Artificial Synthesis

<400>16

aaacatcacc atcaccatca cccca 25

<210>17

<211>85

<212>DNA

<213> Artificial Synthesis

<400>17

tgccaacgac ggcatttgaa agaaccacca ccaccagaac caccaccacc agaaccacca 60

ccacccggca gtacccgctg gtagg 85

Claims

1. A preparation method of bioactive small peptide based on protein scaffold is characterized by comprising the following steps: according to the characteristics of the Titin protein and the Telethion protein, the recombinant plasmid is respectively connected with LfcinB in series to construct pET-24a-Titin-LfcinB and pET-24 a-Telethion-LfcinB recombinant plasmids, and two fusion antibacterial peptides of Titin-LfcinB and Telethion-LfcinB are prepared by taking escherichia coli as a host, wherein Titin-LfcinB: mixing the Telethion-LfcinB monomers according to the molar weight ratio of 1-4:1-4 to prepare ZT-LfcinB, wherein 1-4 monomers are stably coupled together in series or in parallel, and Native SDS-PAGE analysis shows that the assembly is successful.

2. The method for preparing the bioactive small peptide based on the protein scaffold as claimed in claim 1, wherein the ratio of Titin-LfcinB: the molar weight ratio of Telothonin-LfcinB is 2: 1.