CN104610457A

CN104610457A - Hybrid antimicrobial peptide as well as encoding gene and application thereof

Info

Publication number: CN104610457A
Application number: CN201510064471.3A
Authority: CN
Inventors: 车媛媛; 查向东; 马利娟; 徐雪姣; 杨晨晨; 恽辉; 余忠丽
Original assignee: ANHUI XIPU BIOTECHNOLOGY CO LTD
Current assignee: ANHUI XIPU BIOTECHNOLOGY CO LTD
Priority date: 2015-02-06
Filing date: 2015-02-06
Publication date: 2015-05-13

Abstract

The invention discloses a hybrid antimicrobial peptide as well as an encoding gene and an application thereof. The hybrid antimicrobial peptide comprises an antimicrobial peptide LfcinB and an antimicrobial peptide G13 which are connected in series, the antimicrobial peptide LfcinB has an amino acid sequence shown in SEQ ID NO.1 and the antimicrobial peptide G13 has an amino acid sequence shown in SEQ ID NO.2; the hybrid antimicrobial peptide has a strong bactericidal activity. The invention also provides a eukaryotic expression system of the hybrid antimicrobial peptide, namely the encoding gene of the hybrid antimicrobial peptide is introduced into a saccharomyces cerevisiae protein expression system; the eukaryotic expression system is easy in operation and purification, safe and nontoxic, simple in process and low in cost, and has a wide application prospect.

Description

A kind of heterozygous antibacterial peptide and encoding gene thereof and application

Technical field

What the present invention relates to is gene engineering technology field, in particular a kind of heterozygous antibacterial peptide and encoding gene thereof and application.

Background technology

At present, because microbiotic is widely used in clinical, serious drug resistance problems is produced.The mankind are badly in need of novel medicine to replace microbiotic, and antibacterial peptide is small molecule Amphiphilic peptide, and length is generally less than 50 amino acid.Derive from the various bacteriums of occurring in nature, plant and animal, widely distributed.Because its Antibacterial Mechanism is unique, not easily produces resistance, and have good water solubility, thermostability is strong, and has a broad antifungal spectrum, is expected to the substitute becoming conventional antibiotic.Along with carrying out in a deep going way of studying antibacterial peptide mechanism of action, molecular structure and functional relationship etc., people are while excavation novel antimicrobial peptide, more focus on finding efficient, that wide spectrum, low toxicity, peptide chain are shorter antibacterial peptide, and T1249 is exactly the such novel antimicrobial peptide of a class.

Antibacterial peptide LfcinB is the size that Bovinelactoferrin discharges from N-end after stomach en-effect is the structural domain that 25 residues form, it is a kind of novel antimicrobial peptide of discovered in recent years, possess antiviral, antitumor, anti-oxidant and regulate the immunity of body and improve enteron aisle to effects such as the absorptions of iron ion, its anti-microbial activity is higher than lactoferrin more than 400 times, has important using value.LfcinB derives from animal itself, aminoacid sequence is FKCRRWQWRMKKLGAPSITCVRRAF, wherein not containing rare amino acid and exogenous chemical components, be a kind of healthy and safe product, LfcinB has nutritional-physiological regulating effect, improve the immunity function of young animal, improve animal intestinal micro-ecology environment, promote the effect of animal health, it can not make bacterial resistance occurred, also can not residue in livestock product, there is the tremendous potential of substitute antibiotics.It has the action effect that conventional antibiotic does not have.The Antibacterial mechanism research display of LfcinB, the Arg residue being positioned at 2 hydrophobic t rp residues of LfcinB side and opposite side 3 positively charged can be combined with bacterium or virocyte film, wherein Trp easily plays a part film steady arm, on positively charged Arg residue and film, the negatively charged ion of phosphoester groups interacts, then interact with the lipopolysaccharides on film, then the hydrophobicity alpha-helix of peptide molecule is made to insert in film by film steady arm Trp, polymerization forms duct, cause rushing down outside content, bacterium or viral kill.The x diffraction analysis of nucleus magnetic resonance (NMR) is utilized to find, the space conformation of LfcinB is the antiparallel beta sheet of distortion, the x diffraction analysis of contrast LF (lactoferrin), the structure of LF is α-helixstructure: can explain just by the conversion of α-helixstructure to beta sheet thus, the bacteriostatic activity of LfcinB is caused to be better than LF, because beta sheet conformation is easier to the surface of bacterium, thus there are more chances contacted with bacterium.

Particle Flow Code (Granu lysin) is the one of cationic antibacterial peptide, from cytotoxic T lymphocyte (CTL) and natural killer cell (NK), is the Major Members of the SAPLIP family that can insert in cytolemma.Research shows, Particle Flow Code can divide lipoid film, the apoptosis of induction mammalian cell, very strong lytic activity is had to Gram-positive and Gram-negative bacterium, fungi, protozoon, tumour cell, infect with series of human, the Clinics and Practices of tumour and immune-mediated disease has and contacts closely.The space structure of Particle Flow Code is formed primarily of five alpha-helixs, and called after H1-H5 structural domain, is connected by loop ring between each structural domain.Find each structural domain anti-microbial activity research, the position with H2 structural domain is the strongest with other structural domain phase specific activities.G13 structural domain is the peptide section fragment containing H2 in Particle Flow Code, containing 19 amino-acid residues, α-helixstructure and loop structure, this peptide section has 5 Arg residues, net charge is+5, can interact with electronegative bacterial membrane component, cause Host Strains membranolysis, can anti-bacteria, fungi activity, but do not affect zooblast and liposome, the more positive charge that antibacterial peptide G13 has and stable both sexes alpha-helix are combined with and help improve fungicidal activity.

The expression system of antibacterial peptide has following source at present: although natural antibacterial peptide wide material sources, output is little in vivo, is difficult to separation and purification, and cost is high.Although the chemosynthesis antibacterial peptide cycle is short, quantities is little, arbitrarily can combine amino-acid residue, there is racemization problem, and production cost is expensive.Genetic engineering technique, mainly through prokaryotic expression and eukaryotic expression approach, has simple to operate, is easy to abstraction and purification, and the feature such as production cost is low, makes it have mass-producing, potentiality that industrialization is produced, becomes study hotspot in the last few years.

Prokaryotic expression system is based on coli expression system, and be grasp expression system the most ripe at present, its advantage is that growth cycle is short, expresses output higher, relative inexpensiveness.But express antibacterial peptide and there is following shortcoming: prokaryotic expression needs smudge cells to discharge antibacterial peptide, adds the difficulty of production cost and separation and purification; Its continuous expression may produce toxic action to host cell, although add fusion head amalgamation and expression can reduce toxicity, need cut fusion head, complex steps; Prokaryotic expression system post translational processing modifies system imperfection, and the biological activity of expression product is lower.

The advantage that the growth of yeast expression system existing prokaryotic organism is fast, simple to operate, has again eukaryote to carry out the advantage of posttranslational modification to protein.Yeast saccharomyces cerevisiae genetic background is clear, easy to operate; Closely, safety, without toxin, can not constitute a threat to biological and environment for yeast saccharomyces cerevisiae and daily life; Protein translation post-treatment can be carried out; Can secreting, expressing be carried out, be easy to purifying; The features such as the simple cost of technique is lower.Yeast saccharomyces cerevisiae can the supernatant of thalline in substratum and abduction delivering as host expresses antibacterial peptide, directly as difunctional fodder additives, both can supplement feed albumen, the conventional antibiotic added in feed can be substituted again, be a kind of new environment-protecting feed additive, there is significant application value.Report at present about yeast saccharomyces cerevisiae secreting, expressing antibacterial peptide is little, and the eukaryotic expression research of present method to antibacterial peptide is significant.

Summary of the invention

The object of the invention is to overcome the deficiencies in the prior art, provide a kind of heterozygous antibacterial peptide and encoding gene thereof and application, to provide, a kind of anti-microbial activity is strong, output is large, cost is low, be easy to the novel antimicrobial peptide of abstraction and purification and eukaryotic expression system thereof.

The present invention is achieved by the following technical solutions:

The invention provides a kind of heterozygous antibacterial peptide, described heterozygous antibacterial peptide comprises the antibacterial peptide LfcinB and antibacterial peptide G13 that are connected in series, described antibacterial peptide LfcinB has the aminoacid sequence as shown in SEQ ID NO:1, and described antibacterial peptide G13 has the aminoacid sequence as shown in SEQ ID NO:2.

Present invention also offers the encoding gene of above-mentioned heterozygous antibacterial peptide, described encoding gene comprises antibacterial peptide LfcinB gene and antibacterial peptide G13 gene.

Described antibacterial peptide LfcinB gene has the nucleotide sequence as shown in SEQ ID NO:3, and described antibacterial peptide G13 gene has the nucleotide sequence as shown in SEQ ID NO:4.

Present invention also offers a kind of recombinant plasmid of encoding gene of above-mentioned heterozygous antibacterial peptide, described recombinant plasmid is the Saccharomyces cerevisiae protein expression vector of the encoding gene containing heterozygous antibacterial peptide.

Described Saccharomyces cerevisiae protein expression vector is pYES2/CT/ α-factor carrier.

Present invention also offers a kind of genetically engineered bacteria containing above-mentioned recombinant plasmid, described genetically engineered bacteria is the brewing yeast cell containing described recombinant plasmid.

Described brewing yeast cell is yeast saccharomyces cerevisiae INVSC1 cell.

Present invention also offers the application of a kind of above-mentioned genetically engineered bacteria in preparation antibacterials.

The present invention has the following advantages compared to existing technology: the invention provides a kind of heterozygous antibacterial peptide and encoding gene thereof and application, this heterozygous antibacterial peptide comprises the antibacterial peptide that antibacterial peptide LfcinB and antibacterial peptide G13 two kinds has different structure feature, the more positive charge had due to antibacterial peptide G13 and stable both sexes alpha-helix, the overall activity of antibacterial peptide can be improved, simultaneously in conjunction with the beta sheet of antibacterial peptide LfcinB, this heterozygous antibacterial peptide is made to be easier to the surface of bacterium, thus had more chances contacted with bacterium, both combine, mutually promote, substantially increase the fungicidal activity of antibacterial peptide, present invention also offers the eukaryotic expression system of this heterozygous antibacterial peptide, encoding gene by this heterozygous antibacterial peptide imports in Saccharomyces cerevisiae protein expression system, have express output higher, easy handling, safety non-toxic element, be easy to purifying, advantage that technique is simple, with low cost, have broad application prospects.

Accompanying drawing explanation

Fig. 1 is the agarose gel electrophoresis figure of the encoding gene of heterozygous antibacterial peptide;

Fig. 2 is the structural representation of recombinant plasmid pYES2-α-LG;

Fig. 3 is the Tricine-SDS-PAGE electrophorogram of the heterozygous antibacterial peptide of genetically engineered bacteria abduction delivering;

Fig. 4 is that heterozygous antibacterial peptide is to Escherichia coli and Bacillus subtilis bacteriostatic activity detection figure.

Embodiment

Elaborate to embodiments of the invention below, the present embodiment is implemented under premised on technical solution of the present invention, give detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.

Embodiment 1

The construction process of the heterozygous antibacterial peptide of the present embodiment, comprises the following steps:

1, the acquisition of antibacterial peptide LfcinB gene

The aminoacid sequence of antibacterial peptide LfcinB, as shown in SEQ ID NO:1, is optimized the nucleotide sequence that it is encoded according to yeast saccharomyces cerevisiae codon preference, obtains the nucleotide sequence as shown in SEQ ID NO:3, this nucleotide sequence of synthetic;

Design primer, and arrange as template with the nucleotides sequence of above-mentioned synthetic, carry out pcr amplification, wherein, introduce Xho I restriction enzyme site in upstream primer LfcinB-F1, downstream primer is not containing restriction enzyme site, and be flat end after amplification, described primer sequence is:

LfcinB-F1：5’-CCG CTCGAGAAAAGATTTAAATGTAG-3’

LfcinB-R1：5’-GGTAGATAATGAACACAATCTTCTCGAAAA-3’

The reaction conditions of pcr amplification is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 62 DEG C of annealing 30s, and 72 DEG C extend 1min, after 30 circulations, then continues to extend 10min at 72 DEG C, after the purified recovery of product of amplification, saves backup at being placed in-20 DEG C.

2, the acquisition of antibacterial peptide G13 gene

Antibacterial peptide G13 is the peptide section fragment of the G13 structural domain of Particle Flow Code, its amino acid residue sequence is as shown in SEQ ID NO:2, according to yeast saccharomyces cerevisiae codon preference, the nucleotide sequence that it is encoded is optimized, obtain the nucleotide sequence as shown in SEQ ID NO:4, this nucleotide sequence of synthetic;

Design primer, and arrange as template with the nucleotides sequence of above-mentioned synthetic, carry out pcr amplification, wherein, introduce EcoR I restriction enzyme site in downstream primer G13-R2, upstream primer is not containing restriction enzyme site, and be flat end after amplification, described primer sequence is:

G13-F2：5’-CAAAGATCTGTTTCTAATGCTGCTACTAGAGTTTG-3’

G13-R2:5 '-G gAATTCthe reaction conditions of TTACCATCTAGATCTACCAGTTCTACAAAC-3 ' pcr amplification is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 60 DEG C of annealing 30s, 72 DEG C extend 1min, after 30 circulations, continue again to extend 10min at 72 DEG C, after the purified recovery of product of amplification, save backup at being placed in-20 DEG C.

3, the structure of heterozygous antibacterial peptide LfcinB-G13 gene

Above-mentioned antibacterial peptide LfcinB gene and antibacterial peptide G13 gene are carried out blunt end cloning, first, T4PNK Starch phosphorylase phosphorylation 30min is added in 37 DEG C of water-baths, be placed in 65 DEG C of water-bath 10min again and make enzyme deactivation, after taking out cooling, add T4 ligase enzyme, 30min is connected at being placed in 37 DEG C, be placed in 65 DEG C of water-bath 10min again and make enzyme deactivation, utilize PCR to reclaim test kit and reclaim gene fragment, namely heterozygous antibacterial peptide LfcinB-G13 gene is obtained, electrophorogram after its recovery as shown in Figure 1, wherein: swimming lane M is that DNA Marker D (is followed successively by 2000bp from top to bottom, 1000bp, 750bp, 500bp, 250bp, 100bp), swimming lane 1 is the encoding gene of heterozygous antibacterial peptide, can find out in figure, the fragment length of the product obtained is about 130bp, consistent with the encoding gene segment length of the heterozygous antibacterial peptide of prediction.

4, the structure of secreting, expressing type recombinant plasmid pYES2-α-LG

As shown in Figure 2, above-mentioned heterozygous antibacterial peptide LfcinB-G13 gene and pYES2/CT/ α-factor carrier (purchased from Invitrogen company) are carried out double digestion with XhoI and EcoRI, product is cut glue and is reclaimed after the agarose gel electrophoresis that weight ratio is 1%, 1h is connected with T4 ligase enzyme at 16 DEG C, transformation of E. coli E.coliDH5 α bacterial strain, bacterium colony after conversion is order-checking after bacterium colony PCR verifies, positive bacteria is dropped into row amplification, extract plasmid, namely obtain the pYES2-α-LG recombinant plasmid containing heterozygous antibacterial peptide gene.

5, the foundation of Saccharomyces Serevisiae Expression System

1) yeast saccharomyces cerevisiae INVSc1 competent cell is prepared:

Step one: inoculate the frozen glycerol stock of 30 μ l yeast saccharomyces cerevisiae INVSc1 in the YPD liquid nutrient medium of 3ml, at 30 DEG C 200 turns, concussion activation of spending the night.

Step 2: the yeast saccharomyces cerevisiae INVSc1 bacterium liquid of 500 μ l activated overnight of transferring is in the YPD liquid nutrient medium of 50ml, and 30 DEG C, 200rpm, concussion is cultured to bacterium liquid OD ₆₀₀be about 1.1.

Step 3: 4 DEG C, centrifugal 5min under 1500g rotating speed, collecting cell, with the sterilized water pressure-vaccum suspension cell gently of 50ml precooling.

Step 4: 4 DEG C, centrifugal 5min under 1500g rotating speed, collecting cell, with the sterilized water suspension cell of 25ml precooling.

Step 5: 4 DEG C, centrifugal 5min under 1500g rotating speed, collecting cell, with the sorbyl alcohol pressure-vaccum suspension cell gently of 25ml precooling.

Step 6: 4 DEG C, centrifugal 5min under 1500g rotating speed, collecting cell, with the sorbyl alcohol suspension cell of 2ml precooling.

Step 7: 4 DEG C, centrifugal 5min under 1500g rotating speed, collecting cell, with the sorbyl alcohol suspension cell of 200 μ l precoolings, is dispensed in the EP pipe of two pipe 2ml, often pipe 100 μ l, is yeast saccharomyces cerevisiae INVSc1 competent cell.

2) transform:

Step one: the pYES2-α-LG recombinant plasmid drawing 10 μ l joins in 100 μ l yeast saccharomyces cerevisiae INVSc1 competent cells, mixes, adds in the electric shock cup of precooling;

Step 2: place 5min on ice;

Step 3: adopt Bio-red company electricity conversion instrument, preset PIC program according to the yeast saccharomyces cerevisiae parameter that electric conversion instrument is recommended, then shock by electricity;

Step 4: the Sorbitol Solution USP adding 500 μ l precoolings immediately in electric shock cup, mixes;

Step 5: by the whole sucking-off of solution in electric shock cup, be evenly applied on yeast uracil-deficient type minimum synthetic medium SC-U flat board;

Step 6: be placed in 30 DEG C of constant incubators, until grow bacterium colony;

3) screen:

Picking 5 single bacterium colonies from SC-U flat board, carry out colony PCR amplification, wherein, the reaction conditions of pcr amplification is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 62 DEG C of annealing 30s, 72 DEG C extend 1min, after 30 circulations, continue again to extend 10min at 72 DEG C, 1.5% (weight) agarose gel electrophoresis qualification amplified production, filters out positive bacterium colony, is the yeast saccharomyces cerevisiae genetically engineered bacteria of described heterozygous antibacterial peptide.

6, the expression of heterozygous antibacterial peptide yeast saccharomyces cerevisiae genetically engineered bacteria

1) induce engineering bacterium expression heterozygous antibacterial peptide structural domain, concrete steps are as follows:

Step one: 200 μ l antibacterial peptide saccharomyces cerevisiae engineered yeasts are forwarded in the SC-U liquid nutrient medium of 20ml, 30 DEG C, 200rpm shaking culture, activated overnight;

Step 2: the OD600 value Y measuring bacterium liquid, by formula X=(Y × 20)/0.4, calculates the OD of bacterium liquid ₆₀₀be diluted to the SC-U inducing culture base unit weight X needed for 0.4, described SC-U inducing culture is that the inductor semi-lactosi adding 4% (weight) in SC-U liquid nutrient medium prepares;

Step 3: 4 DEG C, centrifugal 5min under 1500g rotating speed, abandon supernatant, the X value calculated by above-mentioned steps two adds SC-U inducing culture, and pressure-vaccum suspends, and mixes;

Step 4: 30 DEG C, 200rpm shaking culture 3 ~ 7 days, gets 1ml bacterium liquid, 4 DEG C, centrifugal 5min under 1500g rotating speed, and obtain supernatant, be induction supernatant, described heterozygous antibacterial peptide is present in this supernatant;

Step 5: get 1ml supernatant, adds the 100%TCA solution of 20 DEG C of precoolings of 5ml, 2h at being then placed in-20 DEG C, and described 100%TCA solution is for formulated by adding 100mL water in 500g trichoroacetic acid(TCA) TCA;

Step 6: 4 DEG C, centrifugal 10min, gently supernatant discarded under 12000rpm, precipitation is placed in ventilation natural air drying, is the heterozygous antibacterial peptide product of expression.

7, heterozygous antibacterial peptide purity detecting

Adopt the purity of heterozygous antibacterial peptide in the induction supernatant of Tricine-SDS-PAGE method qualification above-mentioned steps 6, obtain electrophorogram as shown in Figure 3, wherein: swimming lane M is that Protein Marker (is followed successively by 66kDa from top to bottom, 45kDa, 35kDa, 27kDa, 20kDa, 14.4kDa, 9.5kDa, 6.5kDa, 4.1kDa), swimming lane 1 be 25 μ l do not induce supernatant, swimming lane 2 is the induction supernatant of 25 μ l, obviously can find out swimming lane 2 there is the band of a treaty 6.0kDa, is heterozygous antibacterial peptide of the present invention in figure.

8, the Activity determination of heterozygous antibacterial peptide structural domain

E.coli DH5 α intestinal bacteria and subtilis are placed in 37 DEG C respectively, incubated overnight under 180rpm, measure the OD of bacterium liquid ₆₀₀value, and be diluted to 0.5 Maxwell unit;

Draw 200 μ L bacterium liquid, be spread evenly across and do not have on antibiotic LB solid medium flat board, place Oxford cup gently on flat board;

Induction supernatant 200 μ L in aspiration step 6, adds in the cup of Oxford, and 200 μ L sterilized waters are set, 200 μ L do not induce supernatant, 10 μ g penbritins are control group, be placed in 37 DEG C of constant incubator incubated overnight, next day observes fungistatic effect;

As shown in Figure 4, in figure: 4A is for induction supernatant is to colibacillary fungistatic effect figure, 4B is that induction supernatant is to the fungistatic effect figure of subtilis, 1 is penbritin control group, 2 is the induction supernatant group that engineering bacteria induces 96h, 3 for the engineering bacteria of not inducing centrifugal after supernatant, 4 is sterilized water; As can be seen from the figure, induction supernatant of the present invention all shows obvious bacteriostatic activity to intestinal bacteria and subtilis, and what heterozygous antibacterial peptide of the present invention was described has obvious antibacterial effect.

Claims

1. a heterozygous antibacterial peptide, it is characterized in that, described heterozygous antibacterial peptide comprises the antibacterial peptide LfcinB and antibacterial peptide G13 that are connected in series, described antibacterial peptide LfcinB has the aminoacid sequence as shown in SEQ ID NO:1, and described antibacterial peptide G13 has the aminoacid sequence as shown in SEQ ID NO:2.

2. an encoding gene for heterozygous antibacterial peptide as claimed in claim 1, is characterized in that, described encoding gene comprises antibacterial peptide LfcinB gene and antibacterial peptide G13 gene.

3. the encoding gene of a kind of heterozygous antibacterial peptide according to claim 2, it is characterized in that, described antibacterial peptide LfcinB gene has the nucleotide sequence as shown in SEQ ID NO:3, and described antibacterial peptide G13 gene has the nucleotide sequence as shown in SEQ ID NO:4.

4. a recombinant plasmid for the encoding gene containing heterozygous antibacterial peptide as claimed in claim 3, it is characterized in that, described recombinant plasmid is the Saccharomyces cerevisiae protein expression vector of the encoding gene containing heterozygous antibacterial peptide.

5. a kind of recombinant plasmid containing heterozygous antibacterial peptide gene according to claim 4, it is characterized in that, described Saccharomyces cerevisiae protein expression vector is pYES2/CT/ α-factor carrier.

6., containing, for example a genetically engineered bacteria for the arbitrary described recombinant plasmid of claim 4 or 5, it is characterized in that, described genetically engineered bacteria is the brewing yeast cell containing described recombinant plasmid.

7. a kind of genetically engineered bacteria according to claim 6, is characterized in that, described brewing yeast cell is yeast saccharomyces cerevisiae INVSc1 cell.

8. the application of genetically engineered bacteria as claimed in claim 6 in preparation antibacterials.