CN103224940A - Blunt snout bream beta-defensin gene and protein encoded by the same - Google Patents

Abstract

The invention discloses blunt snout bream beta-defensin gene, which has a nucleotide sequence represented by SEQ ID NO:1. The invention further discloses recombinant protein encoded by the blunt snout bream beta-defensin gene, and a preparation method thereof, wherein the recombinant protein has an amino acid sequence represented by SEQ ID NO:2. The blunt snout bream beta-defensin recombinant protein provides good bacterial inhibition activities for staphylococcus aureus, streptococcus, ampicillin resistance type escherichia coli, aeromonas hydrophila and the like, and provides a class of new drugs with good therapeutic effects for anti-infective drugs and drug-resistant bacteria. The preparation method for the blunt snout bream beta-defensin recombinant protein has the following advantages that: high purity products can be easily obtained; and compared to the existing chemical synthesis method, the method of the present invention has characteristics of good activity, cost reduction, preparation time shortening, and easy industrial production achievement.

Description

Megalobrama amblycephala beta-defensin gene and encoded protein thereof

Technical field

The invention belongs to gene engineering technology field, relate to a kind of new natural antibacterial peptide, specifically be meant a kind of Megalobrama amblycephala beta-defensin gene and encoded protein and application.

Background technology

Bacterium has become the great public health problem that the whole world is paid close attention to the antibiotic resistance of tradition.WHO is described as " influencing the global publilc health emergency case of All Countries " with the microbiotic tolerance, and therefore the ratio that this problem causes global health problem, develops the new antibiotic that overcomes the resistance problem and seem more and more important in increase gradually.The antibacterial peptide of Chu Xianing is exactly the huge novel anti mushroom medicine of a class development potentiality in recent years, it not only has the wide spectrum killing action to bacterium and fungi, but also has well antiviral and antitumor action, in addition, compare with traditional microbiotic, its mechanism of action uniqueness is difficult for causing the resistance of microorganism, the overwhelming majority does not damage or destroys the normal cell of higher animal, so it very likely becomes the newtype drug that alternative clinically antibiosis is usually treated infectation of bacteria.

Compare terrestrial animal and higher mammal, the antibacterial peptide kind of fish is abundanter, also has more uniqueness, is the huge treasure-house of also effectively not developed in the antibacterial peptide Biological resources.Existing report confirms that some antibacterial peptide of fish has stronger fungicidal activity than high vertebrates, therefore, the development and use research of fish antibacterial peptide is had important scientific meaning and actual application value.Alexin is one group of little cationic antibacterial peptide, difference according to cysteine residues position and disulfide linkage mode of connection, but α, β, three kinds of alexins of θ, wherein (β-defensin) each organizes and mucous epithelium beta-defensin in body because of wide expression, is the first line of defence that the host resists extraneous pathogenic micro-organism invasion, is considered to most important alexin.But natural alexin source is limited, and also there is the cost height in chemosynthesis, produces difficulty in batches; The space structure of alexin anti-microbial activity and peptide molecule is closely related, though external synthetic alexin is consistent with natural alexin primary structure, space structure is not quite similar, thereby causes active relatively poor.Therefore, adopting gene engineering method to prepare alexin has great advantage tool.Pichia pastoris phaff (Pichia pasoris) is owing to have the translation post-treatment of good operability of prokaryotic cell prokaryocyte and eukaryotic system, the characteristics of modification concurrently, and fermentation condition is simple, the expression level height is fit to high-density culture, for industrialization and purifying provide great convenience.

Summary of the invention

First purpose of the present invention is to obtain the beta-defensin gene from Megalobrama amblycephala.

Second purpose of the present invention is that described Megalobrama amblycephala beta-defensin gene is efficiently expressed the acquisition recombinant expression protein in yeast expression system.

The 3rd purpose of the present invention provides Application of Recombinant.

(1) acquisition of Megalobrama amblycephala beta-defensin gene of the present invention: the method for utilizing reverse transcription-polymerase chain reaction (RT-PCR), with the Megalobrama amblycephala liver total RNA is template, obtain Megalobrama amblycephala beta-defensin gene through RT-PCR, the open reading frame of its cDNA sequence (ORF) is shown in sequence table SEQ ID NO:1.

(2) the encoded recombinant protein of described Megalobrama amblycephala beta-defensin gene, its aminoacid sequence is shown in sequence table SEQ ID NO:2.

(3) described Megalobrama amblycephala beta-defensin recombinant protein (or polypeptide), its preparation method is: with sequence table SEQ ID NO:1 described Megalobrama amblycephala beta-defensin gene open reading frame (ORF)) make up recombinant expression plasmid pPICZ α-BD1 with Yeast expression carrier, electricity is converted among the Pichia yeast engineering GS115, microbiotic Zeocin screens the transformant of high resistance, and expresses with methanol induction.Collect the supernatant liquor that above-mentioned methanol induction is expressed, its albumen or polypeptide that carries out separation and purification is Megalobrama amblycephala beta-alexin 1 recombinant protein (or polypeptide).

(4) the Megalobrama amblycephala beta-defensin recombinant protein biologically active of the present invention's acquisition has anti-microbial effect.Adopting the Oxford agar diffusion method that the yeast expressed supernatant liquor of recombinating is carried out the bacteriostatic activity determination experiment proves: Megalobrama amblycephala beta-defensin recombinant protein all has good bacteriostatic activity to part gram-positive microorganism such as streptococcus aureus, streptococcus agalactiae and part Gram-negative bacteria such as intestinal bacteria and Aeromonas hydrophila etc., and Megalobrama amblycephala beta-defensin recombinant protein is obvious to the Aeromonas hydrophila and the intestinal bacteria bacteriostatic action of ammonia benzyl drug-resistant type.Therefore, Megalobrama amblycephala beta-defensin of the present invention can be applicable to prepare disease-resistant becteriums product, and disease-resistant becteriums product comprises pharmaceutical prod such as antibacterials, fodder additives, veterinary drug, sanitas etc.

Beneficial effect of the present invention:

The Megalobrama amblycephala beta-defensin recombinant protein that the present invention relates to all has good bacteriostatic activity to streptococcus aureus, suis and ammonia benzyl drug-resistant type intestinal bacteria and Aeromonas hydrophila etc., for anti-infectives and drug-resistant type bacterium provide a class eutherapeutic new medicine.The preparation method of Megalobrama amblycephala beta-defensin recombinant protein provided by the present invention not only is easy to obtain the high product of purity, and active better with respect to existing chemical synthesis process, reduce cost, shorten preparation time, help suitability for industrialized production.

Description of drawings

Fig. 1 is a Megalobrama amblycephala liver total RNA electrophoresis result.

Fig. 2 Megalobrama amblycephala beta-defensin of the present invention gene RT-PCR amplified production.1: goal gene; M:DNA molecular weight standard (precious biological) available from Dalian.

Fig. 3 is the product that primer amplification Megalobrama amblycephala beta-defensin gene is expressed in utilization of the present invention.1: goal gene; M:DNA molecular weight standard (precious biological) available from Dalian.

Fig. 4 is for being that Megalobrama amblycephala beta-defensin recombinant protein is measured the streptococcus aureus bacteriostatic activity among the present invention.1: penbritin 10 μ g; 2:pPICZ α A yeast transformant induced product; 3:pPICZ α A-beta-alexin transformant induced product I; 4:pPICZ α A-beta-alexin transformant induced product II.

Fig. 5 is for being that the recombinant beta alexin is measured the Aeromonas hydrophila bacteriostatic activity among the present invention.1: penbritin 10 μ g; 2:pPICZ α A yeast transformant induced product; 3:pPICZ α A-BD1 transformant induced product I; 4:pPICZ α A-BD1 transformant induced product II.

Fig. 6 measures the streptococcus agalactiae bacteriostatic activity for recombinant protein among the present invention.1: penbritin 10 μ g; 2:pPICZ α A yeast transformant induced product; 3:pPICZ α A-BD1 transformant induced product I; 4:pPICZ α A-BD1 transformant induced product II.

Fig. 7 measures for recombinant protein intestinal bacteria bacteriostatic activity among the present invention.1: penbritin 10 μ g; 2:pPICZ α A yeast transformant induced product; 3:pPICZ α A-BD1 transformant induced product I; 4:pPICZ α A-BD1 transformant induced product II.

Fig. 8 is a nucleotide sequence of the present invention.

Fig. 9 is an aminoacid sequence of the present invention.

The aminoacid sequence comparison chart of the beta-defensin that derives from different plant species that Figure 10 has announced for NCBI.Black box is a conserved sequence.

Figure 11 is for inserting the nucleotide sequence of expression vector among the present invention.

Embodiment

The present invention is described in detail below in conjunction with embodiment.

Embodiment 1: the extraction of the total RNA of Megalobrama amblycephala and cDNA's is synthetic

1) extraction of the total RNA of Megalobrama amblycephala: the glassware of experiment usefulness is through 0.1% DEPC water treatment, 150 ℃ of baking 4h.Plastic ware spends the night 121 ℃ of 20min autoclave sterilizations through 0.1% DEPC water logging bubble.The metal apparatus soaks 2h through the NaOH of 1mol/L, behind 0.01% DEPC water cleaning down, and 37 ℃ of oven dry.(Ribonuclease, 0.01% DEPC water RNase) disposes each reagent with deoxyribonuclease.MS222 anaesthetizes Megalobrama amblycephala with narcotic, dissects, and takes out liver, and clip 0.1g liver extracts RNA.Total RNA extracting is undertaken by the specification sheets of Trizol reagent.The RNA that extracts carries out 1.5% agarose gel electrophoresis, uses the gel imaging instrument to observe and identifies, as seen three bands (Fig. 1) clearly.Total RNA sample is frozen in-80 ℃, standby.

2) cDNA(reverse transcription product) article one chain is synthetic: the Megalobrama amblycephala liver total RNA with extraction is that template is carried out reverse transcription, and reaction system is 40 μ L, that is:

No RNase water 8 μ L,

Total RNA15 μ L,

Oligo dT(18)-Adaptor primer 2 μ L,

70 ℃ following 10 minutes, ice bath cooling immediately adds respectively afterwards:

5 * RT damping fluid, 8 μ L,

DNTP mixed solution 4 μ L,

RNase inhibitor 1.5 μ L,

ThermoScript II 1.5 μ L,

Amount to 40 μ L,

Mixing, the reverse transcription reaction condition is: 42 ℃ following 20 minutes, 70 ℃ following 15 minutes, last 4 ℃ are finished reaction, 4 ℃ of preservations are standby.

Embodiment 2: the clone and the sequential analysis of Megalobrama amblycephala beta-defensin gene

1) design of primers

According to having logined fish beta-defensin sequence information on the NCBI/GenBank, according to the conservative property of sequence, with Primer 5.0 softwares in the upstream and downstream zone of cDNA sequence open reading frame (ORF), design a pair of primer (F1, R1); F1:5 '-GCCATCATCCGAAGAAAC-3 '; R1:5 '-TTCCAAATCAAAGGCATG-3 '.

2) pcr amplification: with reverse transcription product (cDNA) is that template is carried out pcr amplification, and reaction system is 50 μ L, that is: Premix Taq 2.025 μ L; Upstream primer F1(100mM) 0.5 μ L; Downstream primer R1(100mM) 0.5 μ L; Reverse transcription product (cDNA) 2 μ L; Sterilization deionized water 22 μ L, cumulative volume is 50 μ L, mixing.The pcr amplification reaction condition is: pre-sex change 3min under 94 ℃; Carry out 30 circulating reactions then, its temperature cycle condition is 94 ℃ of following sex change 30s, 48 ℃ of 30s that anneal down, and 72 ℃ are extended 1min down; Under 72 ℃, extend 10min after the loop ends again.

3) the PCR product is identified: after amplification is finished, get PCR product 5 μ L with 6 * nucleic acid sample-loading buffer point sample, and 1.0% sepharose, 1 * TAE damping fluid, 120V, 20 minutes observationss of electrophoresis obtain the PCR product (see figure 2) about about 334bp.

4) clone of goal gene, screening and order-checking

Get pMD19-T carrier 1 μ L(50ng/uL) with after purpose fragment 3 μ L that glue reclaims mix, add 6 μ L solution I, 16 ℃ of connections of mixing postposition are spent the night.Get 10 μ L and connect product, aseptic condition adds in the intestinal bacteria TOP10 competent cell down, blows and beats mixing repeatedly with the pipettor gentleness, and ice bath is placed 30min.42 ℃ of water-baths, heat shock 90s, ice bath 3min makes it cooling immediately afterwards, notes not rocking.Shift bacterium liquid to being equipped with in the LB nutrient solution that 500 μ L are preheated to 37 ℃, 37 ℃ of gentle vibration 45min of 150rpm make bacterium recover resistance.On the LB agar plate that contains penbritin (AMP) (100 μ g/mL), add 40 μ L X-gal (20mg/mL), 4 μ L IPTG (200mg/mL), evenly coating, 37 ℃ place half an hour after, get 150 μ L bacterium liquid and coat on the flat board.Be inverted plate and cultivate 12 ~ 16h, place 4 ℃ blueness is fully manifested, choose white colony and be inoculated in the LB nutrient solution that contains 150 μ g/mL AMP, identify behind violent jolting (230rpm) 12 ~ 16h in 37 ℃ of constant incubators.The clone that pcr amplification is positive, getting bacterium liquid send China big gene biological engineering service company, carry out sequencing analysis, obtain 334bp(and see Fig. 8) the cDNA sequence, this gene has complete open reading frame (ORF), the sequence of the sequence of its open reading frame shown in sequence table SEQ ID NO:1.

Sequence number: SEQ ID NO:1

Sequence length: 204bp

Sequence type: cDNA

Source: Megalobrama amblycephala

Sequence signature: correct open reading frame (ORF): 204bp is arranged; Decision position: initial, terminator codon location: ATG, 1; TGA, 204

5) 4 calling sequence ORF of above-mentioned steps total length 204bp, coded protein has the aminoacid sequence shown in sequence table SEQ ID NO:2.This protein contains 67 amino acid, and molecular weight is 7.17KD, contain 4 strong basicity amino-acid residues (K, R), 3 strongly-acid amino-acid residues (D, E), 29 hydrophobic amino acid residues (A, I, L, F, W, V), 18 polare Aminosaerens (N, C, Q, S, T, Y).Utilize the American National biotechnology (NCBI of information center, National Center for Biotechnology Information, http: the BLAST(Basic Local Alignment Search Tool of website // www.ncbi. nlm.nih.gov)) software is analyzed the survey aminoacid sequence.With the Megalign software in the DNAstar software package, by nucleotide sequence similarity (see figure 10) between the ClustalW method different plant species relatively.The result shows the total length ORF coding region sequence that has obtained Megalobrama amblycephala beta-defensin gene.

Embodiment 3: the structure of recombinant eukaryon expression vector and the abduction delivering in pichia spp

1) the expression primer of design Megalobrama amblycephala beta-defensin:

According to Megalobrama amblycephala beta-defensin ORF sequences Design express primer (F2, R2), upstream primer F2 removes 5 ' signal peptide sequence, and adding EcoR I restriction enzyme site, downstream primer add terminator codon (TGA, annotate: complementary codon TCA) and Xba I restriction enzyme site, specific as follows:

F2：5' CG GAATTC ATACTTGTCTTGCTTGTCC 3'

EcoRⅠ

R2： 5' GC TCTAGATCA ATGTGATACACAGCATAAG 3'

XbaⅠ

2) structure of recombinant expression plasmid

With expressing primer (F2, R2) recombinant plasmid is carried out the pcr amplification (see figure 3), purpose fragment and expression vector pPICZ α A use restriction enzyme EcoR I and Xba I to carry out the substep double digestion, 16 ℃ of connections of T4 ligase enzyme are spent the night, transformed into escherichia coli TOP10, make up eukaryotic expression recombination plasmid pPICZ α A-beta-alexin, order-checking is identified through the big genome company of China, determines that this gene correctly is inserted among the expression vector pPICZ α A (seeing Figure 11).

3) linearizing of recombinant expression plasmid

Correct recombinant expression plasmid pPICZ α A-beta-alexin and the pPICZ α A empty carrier of order-checking cut with restriction enzyme BstX I enzyme respectively, and system is as follows:

Deionized water 10 μ L,

Plasmid 30 μ L,

10 * H damping fluid, 5 μ L,

BstX I5μL，

Amount to 50 μ L,

45 ℃ of reaction 5h, enzyme is cut product and is carried out purifying with DNA cleaning agents box, and 20 μ L deionized waters carry out wash-out ,-20 ℃ of preservations.

4) recombinant plasmid is imported the screening of yeast competent cell and positive yeast colony

With pichia spp (Pichia pastoris) GS115 competent cell 80 μ L and pPICZ α A-beta-alexin plasmid (the 20 μ L) mixing after BstX I linearizing, be transferred in the 0.2cm electricity revolving cup (Bio-Red) of precooling, place 5min on ice, 2kV, 25 μ F, 400 Ω shock by electricity after 8 milliseconds, the 1mol/L sorbyl alcohol that adds the 1mL precooling is immediately got 200 μ L separate application in containing uncle's Lay mycin (Zeocin ^TM) on the YPDS flat board of (100 μ g/mL, 500 μ g/mL, 1000 μ g/mL), cultivate 72h for 28 ℃, be cultured to single bacterium colony and occur.Electricity transforms the pPICZ α A empty carrier that does not add any foreign gene simultaneously.

After the positive bacterium colony of picking is used YPD+ZeocinTM 250rpm shaking culture, adopt to boil and freeze method extraction recombination yeast genomic dna.With the centrifugal 5min of 1mL bacterium liquid 2500g, abandon supernatant, add 500 μ L TE damping fluids suspension precipitation in the precipitation; The centrifugal 3min of 2500g abandons supernatant; Repeat step once; Precipitation is dissolved in 100 μ L TE, boiling water bath 10min ,-80 ℃ of freezing 30min, boiling water bath 10min once more at last; The centrifugal 5min of 1500g; Getting supernatant 2 μ l is that template is PCR.

With the recombination yeast genome is that template is carried out pcr amplification, and reaction system is 20 μ L, that is:

Premix Taq 2.010μL，

Upstream primer 5 ' AOX (100 mM) 0.5 μ L,

Downstream primer 3 ' AOX (100 mM) 0.5 μ L,

Template 1 μ L,

Sterilization deionized water 8 μ L,

Amount to 20 μ L,

Mixing.The pcr amplification reaction condition is: pre-sex change is 3 minutes under 94 ℃; Carry out 30 circulating reactions then, its temperature cycle condition is 94 ℃ of following sex change 30 seconds, anneals 30 seconds down for 48 ℃, and 72 ℃ were extended 45 minutes down; Under 72 ℃, extended again 10 minutes after the loop ends.

After amplification is finished, get PCR product 5 μ L with 6 * nucleic acid sample-loading buffer point sample, 1.5% sepharose, 1 * TAE damping fluid, 120V, 20 minutes observationss of electrophoresis.

5) abduction delivering of high copy positive strain

Select PCR to be accredited as male recombination yeast bacterium colony and be inoculated in 250 milliliters of Erlenmeyer flasks that contain 20mL BMGY substratum, 28 ℃, 250 rotational oscillations swing to be cultivated 12 ~ 16 hours, made OD600=2 ~ 6, and centrifugal collection thalline discards the BMGY substratum.An amount of thalline is transferred in 1000 milliliters of Erlenmeyer flasks that contain 100mL BMMY substratum, 4 layers of gauze wrapping bottleneck, making initial OD600 is about 1.At 28 ℃, begin methanol induction under the 250 commentaries on classics conditions and express.Every 24 hours, the adding final concentration was 0.8% methyl alcohol in Erlenmeyer flask, and replenishes an amount of sterilized water, induces pPICZ α A empty carrier yeast simultaneously.

Embodiment 4: the bacteriostatic activity of Megalobrama amblycephala beta-defensin recombinant protein (or polypeptide) is identified

Behind the methanol induction 72 hours, 10000 leave the heart collects the expression supernatant, 0.45 μ m membrane filtration is expressed supernatant, stirs on ice to add solid ammonium sulfate, making saturation ratio is 85%, after ammonium sulfate dissolves fully, 4 ℃ of standing over night, 14000 changeed Superfreezing centrifugal 30 minutes, abandoned supernatant, with 5mL PBS solution dissolution precipitation, 4 ℃ of preservations.Same method concentrates the yeast expressed supernatant of pPICZ α A empty carrier.

Adopting the Oxford agar diffusion method that the yeast expressed supernatant liquor of recombinating is carried out bacteriostatic activity measures: line streptococcus aureus and intestinal bacteria on the LB substratum, line streptococcus agalactiae and Aeromonas hydrophila are cultured to mono-clonal and grow on the BHI substratum.With 0.65% stroke-physiological saline solution dilute bacterial strain to be measured to OD600 be 0.5 ~ 1, draw 50 μ L bacterium liquid to be measured add to the liquid LB that is cooled to below 50 ℃ and BHI solid medium and in, fall dull and stereotyped behind the mixing.On the flat board that solidifies, place sterilized Oxford cup, in the cup of Oxford, add 100 μ L concentrate 20 times the abduction delivering supernatant (if no special instructions, specimen in use all is to concentrate 20 times supernatant, slightly following), to add equal volume AMP (0.1mg/mL), concentrate the negative contrast of the yeast expressed supernatant of pPICZ α A empty carrier.Be cultured to thalline and grow, observe the inhibition zone size.Streptococcus aureus and intestinal bacteria culture temperature are 37 ℃, and streptococcus agalactiae, Aeromonas hydrophila culture temperature are 28 ℃.Antibacterial effect is seen accompanying drawing 4 ~ 7.The result shows that Megalobrama amblycephala beta-defensin recombinant protein has good bacteriostatic activity to gram-positive microorganism streptococcus aureus (see figure 4) and streptococcus agalactiae (see figure 6) and Gram-negative bacteria Aeromonas hydrophila (see figure 5), intestinal bacteria (see figure 7), wherein penbritin does not have bacteriostatic action (ammonia benzyl drug-resistant type) to Aeromonas hydrophila and intestinal bacteria, and Megalobrama amblycephala beta-alexin 1 recombinant protein is obvious to the Aeromonas hydrophila and the intestinal bacteria bacteriostatic action of ammonia benzyl drug-resistant type.

Claims (5)

1. Megalobrama amblycephala beta-defensin gene, this gene has the nucleotide sequence shown in SEQ ID NO:1.

2. the recombinant protein of coding claim 1 described Megalobrama amblycephala beta-defensin gene, this recombinant protein has the aminoacid sequence shown in sequence table SEQ ID NO:2.

3. the preparation method of the described recombinant protein of claim 2 is characterized in that:

The gene recombination of the described Megalobrama amblycephala beta-defensin of sequence table SEQ ID NO:1 to Yeast expression carrier, is obtained recombinant protein behind the abduction delivering.

4. the application of the described recombinant protein of claim 2 in the disease-resistant mushroom medicine of preparation, fodder additives, veterinary drug, sanitas.

5. the application of the described recombinant protein of claim 2 in the anti-streptococcus aureus of preparation, streptococcus agalactiae, Aeromonas hydrophila, colibacillary medicine, fodder additives, veterinary drug, sanitas.

Images