CN103224940B - 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

The albumen of Megalobrama amblycephala beta-defensin gene and coding thereof

Technical field

The invention belongs to gene engineering technology field, relate to a kind of new natural antibacterial peptide, specifically refer to albumen and the application of a kind of Megalobrama amblycephala beta-defensin gene and coding thereof.

Background technology

Bacterium has become the great public health problem of global concern to the antibiotic resistance of tradition.Microbiotic tolerance is described as " affect the global publilc health emergency case of All Countries " by WHO, and this problem causes the ratio of Global Health problem increasing gradually, therefore, develops the new antibiotic that overcomes resistance problem and seem more and more important.The antibacterial peptide occurring is in recent years exactly the huge novel antibacterial class medicine of a class development potentiality, it not only has wide spectrum killing action to bacterium and fungi, but also there is well antiviral and antitumor action, in addition, compared with traditional microbiotic, its mechanism of action uniqueness, is difficult for causing the resistance of microorganism, most do not damage or destroy the normal cell of higher animal, therefore it very likely becomes substitute antibiotics clinically and treats the newtype drug that bacterium infects.

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

Summary of the invention

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

Second object of the present invention is that described Megalobrama amblycephala beta-defensin gene high efficient expression in yeast expression system is obtained to recombinant expression protein.

The 3rd object of the present invention is to provide the application of recombinant protein.

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

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

(3) described Megalobrama amblycephala beta-defensin recombinant protein (or polypeptide), its preparation method is: by the Megalobrama amblycephala beta-defensin gene open reading frame (ORF) described in sequence table SEQ ID NO:1) build recombinant expression plasmid pPICZ α-BD1 with Yeast expression carrier, electricity is converted in 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, albumen or polypeptide that it is carried out to separation and purification are Megalobrama amblycephala beta-alexin 1 recombinant protein (or polypeptide).

(4) the Megalobrama amblycephala beta-defensin recombinant protein that the present invention obtains has biological activity, has anti-microbial effect.Adopting Oxford agar diffusion method to carry out Antibacterial Activity to the yeast expressed supernatant liquor of recombinating experimental results show that: Megalobrama amblycephala beta-defensin recombinant protein all has good bacteriostatic activity as intestinal bacteria and Aeromonas hydrophila etc. to part gram-positive microorganism as streptococcus aureus, streptococcus agalactiae and part Gram-negative bacteria, and Aeromonas hydrophila and the intestinal bacteria bacteriostatic action of Megalobrama amblycephala beta-defensin recombinant protein to ammonia benzyl drug-resistant type is obvious.Therefore, Megalobrama amblycephala beta-defensin of the present invention can be applicable to prepare Anti-bacterium series products, and Anti-bacterium series products comprises that pharmaceutical prod is as antibacterials, fodder additives, veterinary drug, sanitas etc.

Beneficial effect of the present invention:

The Megalobrama amblycephala beta-defensin recombinant protein 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 is not only easy to obtain the product that purity is high, and active better with respect to existing chemical synthesis process, reduce costs, shorten preparation time, be conducive to suitability for industrialized production.

Brief description of the drawings

Fig. 1 is 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 purchased 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 purchased from Dalian).

Fig. 4 for be in the present invention Megalobrama amblycephala beta-defensin recombinant protein to streptococcus aureus Antibacterial Activity.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 for be in the present invention recombinant beta alexin to Aeromonas hydrophila Antibacterial Activity.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 be in the present invention recombinant protein to streptococcus agalactiae Antibacterial Activity.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 is recombinant protein intestinal bacteria Antibacterial Activity in 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 nucleotide sequence of the present invention.

Fig. 9 is aminoacid sequence of the present invention.

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

Figure 11 is the nucleotide sequence that inserts expression vector in the present invention.

Embodiment

Below in conjunction with embodiment, the present invention is described in detail.

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 use is through 0.1% DEPC water treatment, 150 DEG C of baking 4h.Plastic ware spends the night through 0.1% DEPC water soaking, 121 DEG C of 20min autoclave sterilizations.Metal apparatus soaks 2h through the NaOH of 1mol/L, after 0.01% DEPC water cleaning down, and 37 DEG C of oven dry.Each 0.01% DEPC water configuration of deoxyribonuclease (Ribonuclease, RNase) for reagent.With narcotic, MS222 anaesthetizes Megalobrama amblycephala, 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 extracting carries out 1.5% agarose gel electrophoresis, uses gel imaging instrument to observe qualification, as seen three bands (Fig. 1) clearly.Total RNA sample is frozen in-80 DEG C, for subsequent use.

2) cDNA(reverse transcription product) Article 1 chain synthetic: carry out reverse transcription taking the Megalobrama amblycephala liver total RNA of extracting as template, reaction system is 40 μ L, that is:

Without RNase water 8 μ L,

Total RNA15 μ L,

Oligo dT(18)-Adaptor primer 2 μ L,

At 70 DEG C 10 minutes, ice bath was 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,

Mix, reverse transcription reaction condition is: at 42 DEG C 20 minutes, at 70 DEG C 15 minutes, last 4 DEG C were finished reaction, and 4 DEG C of preservations are for subsequent use.

Embodiment 2: 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 NCBI/GenBank, according to the conservative property of sequence, in the upstream and downstream region of cDNA sequence open reading frame (ORF), design pair of primers (F1, R1) with Primer 5.0 softwares; F1:5 '-GCCATCATCCGAAGAAAC-3 '; R1:5 '-TTCCAAATCAAAGGCATG-3 '.

2) pcr amplification: carry out pcr amplification taking reverse transcription product (cDNA) as template, 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; Sterilizing deionized water 22 μ L, cumulative volume is 50 μ L, mixes.Pcr amplification reaction condition is: denaturation 3min at 94 DEG C; Then carry out 30 circulating reactions, its temperature cycle condition is sex change 30s at 94 DEG C, the 30s that anneals at 48 DEG C, 72 DEG C of downward-extension 1min; After loop ends, at 72 DEG C, extend again 10min.

3) PCR Product Identification: after having increased, get PCR product 5 μ L 6 × nucleic acid sample-loading buffer point samples, 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 glue reclaim object fragment 3 μ L mixes after, add 6 μ L solution I, mix postposition 16 DEG C of connections spend the night.Get 10 μ L and connect product, add in intestinal bacteria TOP10 competent cell under aseptic condition, repeatedly blow and beat and mix by pipettor gentleness, ice bath is placed 30min.42 DEG C of water-baths, heat shock 90s, ice bath 3min makes it cooling immediately afterwards, notes not rocking.Shift bacterium liquid to 500 μ L are housed are preheated in the LB nutrient solution of 37 DEG C, 37 DEG C of gentle vibration 45min of 150rpm, make bacterium recover resistance.To adding 40 μ L X-gal (20mg/mL), 4 μ L IPTG (200mg/mL) containing on the LB agar plate of penbritin (AMP) (100 μ g/mL), evenly coating, places after half an hour, gets 150 μ L bacterium liquid and coat on flat board for 37 DEG C.Be inverted plate and cultivate 12 ~ 16h in 37 DEG C of constant incubators, be placed in 4 DEG C blueness is fully manifested, choose white colony and be inoculated in containing in the LB nutrient solution of 150 μ g/mL AMP, after violent jolting (230rpm) 12 ~ 16h, identify.By clone positive pcr amplification, get bacterium liquid and send Hua Da gene biological engineering service company, carry out sequencing analysis, obtain 334bp(and see Fig. 8) cDNA sequence, this gene has complete open reading frame (ORF), the sequence of the sequence of its open reading frame as 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: have correct open reading frame (ORF): 204bp; Determine position: initial, terminator codon location: ATG, 1; TGA, 204

5) 4 calling sequence ORF total length 204bp of above-mentioned steps, coded protein has the aminoacid sequence as shown in sequence table SEQ ID NO:2.This protein contains 67 amino acid, and molecular weight is 7.17KD, contains 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: // www.ncbi. nlm.nih.gov) the BLAST(Basic Local Alignment Search Tool of website) software analyzes surveyed aminoacid sequence.With the Megalign software in DNAstar software package, by nucleotide sequence similarity (see figure 10) between the different plant species of ClustalW method comparison.Result has shown to obtain the total length ORF coding region sequence of 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:

Express primer (F2, R2) according to Megalobrama amblycephala beta-defensin ORF sequences Design, upstream primer F2 removes 5 ' signal peptide sequence, and add EcoR I restriction enzyme site, downstream primer adds terminator codon (TGA, note: 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 to pcr amplification (see figure 3), object fragment and expression vector pPICZ α A use restriction enzyme EcoR I and Xba I to carry out substep double digestion, 16 DEG C of connections of T4 ligase enzyme are spent the night, transform intestinal bacteria TOP10, build eukaryotic expression recombination plasmid pPICZ α A-beta-alexin, through the order-checking qualification of Hua Da genome company, determine that this gene is correctly inserted in 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 DEG C of reaction 5h, enzyme is cut product DNA cleaning agents box and is carried out purifying, and 20 μ L deionized waters carry out wash-out ,-20 DEG C of preservations.

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

Pichia spp (Pichia pastoris) GS115 competent cell 80 μ L are mixed with the pPICZ α A-beta-alexin plasmid (20 μ L) after BstX I linearizing, be transferred in the 0.2cm electricity revolving cup (Bio-Red) of precooling, be placed in 5min on ice, 2kV, 25 μ F, 400 Ω, shock by electricity after 8 milliseconds, add immediately the 1mol/L sorbyl alcohol of 1mL precooling, get 200 μ L and coat respectively 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 DEG C, be cultured to single bacterium colony and occur.Electricity transforms the pPICZ α A empty carrier that does not add any foreign gene simultaneously.

The positive bacterium colony of picking, with after YPD+ZeocinTM 250rpm shaking culture, adopts to boil and freezes method extraction recombination yeast genomic dna.By the centrifugal 5min of 1mL bacterium liquid 2500g, abandon supernatant, in precipitation, add 500 μ L TE damping fluids to suspend and precipitate; The centrifugal 3min of 2500g, abandons supernatant; Repeat step once; Finally precipitation is dissolved in 100 μ L TE, boiling water bath 10min ,-80 DEG C of freezing 30min, again boiling water bath 10min; The centrifugal 5min of 1500g; Getting supernatant 2 μ l is that template is PCR.

Carry out pcr amplification taking recombination yeast genome as template, 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,

Sterilizing deionized water 8 μ L,

Amount to 20 μ L,

Mix.Pcr amplification reaction condition is: denaturation 3 minutes at 94 DEG C; Then carry out 30 circulating reactions, its temperature cycle condition is sex change 30 seconds at 94 DEG C, anneal 30 seconds at 48 DEG C, and 72 DEG C of downward-extensions 45 minutes; After loop ends, at 72 DEG C, extend again 10 minutes.

After having increased, get PCR product 5 μ L 6 × nucleic acid sample-loading buffer point samples, 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 positive recombination yeast bacterium colony and be inoculated in 250 milliliters of Erlenmeyer flasks containing 20mL BMGY substratum, 28 DEG C, 250 turn shaking culture 12 ~ 16 hours, make OD600=2 ~ 6, and centrifugal collection thalline discards BMGY substratum.Appropriate thalline is transferred in 1000 milliliters of Erlenmeyer flasks containing 100mL BMMY substratum, 4 layers of gauze wrapping bottleneck, making initial OD600 is 1 left and right.At 28 DEG C, 250 turn and under condition, start methanol induction and express.Every 24 hours, be 0.8% methyl alcohol to adding final concentration in Erlenmeyer flask, and supplement appropriate sterilized water, induce pPICZ α A empty carrier yeast simultaneously.

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

After methanol induction 72 hours, 10000 leave the heart collects expression supernatant, 0.45 μ m membrane filtration is expressed supernatant, stirs and adds solid ammonium sulfate on ice, and making saturation ratio is 85%, after ammonium sulfate dissolves completely, 4 DEG C of hold over night, 14000 turn Superfreezing centrifugal 30 minutes, abandon supernatant, with 5mL PBS solution dissolution precipitation, 4 DEG C of preservations.The concentrated yeast expressed supernatant of pPICZ α A empty carrier of same method.

Adopt Oxford agar diffusion method to carry out Antibacterial Activity to the yeast expressed supernatant liquor of recombinating: on LB substratum, rule streptococcus aureus and intestinal bacteria, on BHI substratum, rule streptococcus agalactiae and Aeromonas hydrophila, be cultured to mono-clonal and grow.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 be cooled to 50 DEG C of following liquid LB and BHI solid medium and in, after mixing, be down flat plate.On the flat board solidifying, place sterilized Oxford cup, to the abduction delivering supernatant that adds concentrated 20 times of 100 μ L in the cup of Oxford (if no special instructions, specimen in use is all supernatants of concentrated 20 times, lower summary), to add same volume AMP (0.1mg/mL), the concentrated negative contrast of the yeast expressed supernatant of pPICZ α A empty carrier.Be cultured to thalline and grow, observe inhibition zone size.Streptococcus aureus and intestinal bacteria culture temperature are 37 DEG C, and streptococcus agalactiae, Aeromonas hydrophila culture temperature are 28 DEG C.Antibacterial effect is shown in accompanying drawing 4 ~ 7.Result shows that Megalobrama amblycephala beta-defensin recombinant protein has good bacteriostatic activity to gram-positive microorganism streptococcus aureus (seeing Fig. 4) and streptococcus agalactiae (seeing Fig. 6) and Gram-negative bacteria Aeromonas hydrophila (seeing Fig. 5), intestinal bacteria (seeing Fig. 7), wherein penbritin is to Aeromonas hydrophila and intestinal bacteria without bacteriostatic action (ammonia benzyl drug-resistant type), and Aeromonas hydrophila and the intestinal bacteria bacteriostatic action of Megalobrama amblycephala beta-alexin 1 recombinant protein to ammonia benzyl drug-resistant type is obvious.

Claims (4)

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

2. by the recombinant protein of Megalobrama amblycephala beta-defensin coded by said gene described in claim 1, the aminoacid sequence of this recombinant protein is as shown in SEQ ID NO:2.

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

Megalobrama amblycephala beta-defensin gene recombination shown in sequence table SEQ ID NO:1, to Yeast expression carrier, is obtained to recombinant protein after abduction delivering.

Described in claim 2 recombinant protein in the application of preparing in anti-Staphylococcus aureus, streptococcus agalactiae, Aeromonas hydrophila, colibacillary medicine.