CN101906165B - Expression product in series of two fish antibacterial peptide genes and expression method thereof - Google Patents

Abstract

The invention discloses an expression product in series of two fish antibacterial peptide genes and an expression method thereof, relating to the gene engineering technology of marine organism, providing an expression product in series of the two fish antibacterial peptide genes and construction of an expression vector in series of two fish Hepcidin and a preparation method for the expression product in series. In the invention, two fish hepcidin genes are expressed in series by a pET32a prokaryocyte expression vector to realize the soluble expression of two antibacterial peptide hepcidin at the same time; the expression level of two antibacterial peptides in series is over 50mg/L, the soluble expression product in series is purified by a N-terminal His-Tag affinity chromatography, and then cuts off a protein label in series by enterokinase; and the cut mixture is purified by a Ni affinity chromatographic column to obtain a purified Hepcidin expression product in series. The purified expression product has obvious inhibiting effect on Staphylococcus aureus, Aeromonas hydrophila, micrococcus lysodeikticus and the like.

Description

The tandem expression product and the expression method thereof of two kinds of fish antibacterial peptide genes

Technical field

The present invention relates to a kind of halobiontic genetic engineering technique; Especially relate to and a kind of large yellow croaker antibacterial peptide hepcidin (being abbreviated as PC-hepc) and porgy antibiotic peptide hepcidin (being abbreviated as AS-hepc2) gene with anti-microbial activity carried out tandem expression, and carry out the activity identification of vivoexpression product.

Background technology

Antibacterial peptide is the one type of micromolecule polypeptide with broad spectrum antibiotic activity that is produced by animal and plant cells, and its generation and release are the integral parts of body inflammatory reaction, is the important barrier of pathogenic micro-organism invasions such as host defense bacterium, fungi.Hepcidin (liver cecropin) is the antibacterial peptide family of on the conservative site of C end, being rich in cysteine residues of a tool β laminated structure, participates in host's non-specific immunity.2000; Krause etc. have at first found the LEAP-1 (antibacterial peptide of liver expression from human plasma; After be called hepcidin), and detected the anti-microbial activity of hepcidin with this polypeptide of chemosynthesis, the result finds; Hepcidin can suppress the growth of gram-positive microorganism micrococcus luteus, Staphylococcus carnosus, bacillus megaterium, subtilis and Gram-negative bacteria grey neisserial in dose-dependently ground, but intestinal bacteria and pseudomonas fluorescens are not had activity.Park etc. are isolating Hep20 and Hepc25 natural product from human urine; In the in-vitro antibacterial test under 30 μ M concentration to intestinal bacteria (E.coliML35P G-) restraining effect that tool is stronger, a little less than the growth-inhibiting effect to gram-positive microorganisms such as gold-coloured staphylococci and staphylococcus epidermidiss.Relevant report shows that people hepcidin is a kind of antibacterial peptide with broad-spectrum sterilization bacteriostatic action, with other are rich in the same host's of participation of antibacterial peptide of halfcystine natural defence in the body.

The applicant carries out experiment in vitro after through chemosynthesis large yellow croaker hepcidin mature peptide and prokaryotic expression AS-hepc2 and has confirmed that also fish hepcidin has antibacterial and sterilization idiocratic.There is the aminoacid sequence of report Hepcidin variant highly similar between planting; And with evolution that the Lu Sheng Mammals has after senior adaptive immune system compare; Because the complicacy of aquatic environment and direct contact; Fish possibly need multiple not on the same group antibacterial peptide exist, and in innate immune system as staple.Must deepen people to the low understanding that waits the vertebrates immune defence mechanism to the research of Hepcidin antibacterial peptide, the hepcidin antibacterial peptide has good popularization and application prospect as the antibiotic and medicine of potential seawater aquaculture simultaneously.But directly the natural hepcidin antibacterial peptide of chemical process synthetic is extracted or utilized to biological chemistry from fish tissue or secretory product, be difficult to obtain a large amount of antibacterial peptides, and time-consuming expensive.Utilize Protocols in Molecular Biology can clone the antibacterial peptide gene that obtains marine fish; Express engineering bacteria through setting up marine fish hepcidin antibacterial peptide gene; Can be at external mass production antibacterial peptide; Carry out the anti-microbial activity detection and substitute microbiotic adding in the feed, be used for prevention and treatment aquatic animal disease.

Because the hepcidin polypeptide contains 8 halfcystines; Have 4 special disulfide linkage structures; What the hepcidin vivoexpression appeared in the newspapers both at home and abroad is few; The yeast expression of report porgy hepcidin mature peptide gene is arranged, but be only limited to dose-dependent inhibition experiment, also do not illustrate detailed problems such as amount that its supernatant expresses and the antibiotic stability of little peptide intestinal bacteria, Pseudomonas aeruginosa and Aeromonas hydrophila.([3] Zhang H, Yuan QP, Zhu YP such as Zhang; Ma RY.Expression and preparation ofrecombinant hepcidin in Escherichia coli [J] .Protein.Expr.Purif.; 2005,41:409-416) utilize the synthetic people hepcidin nucleic acid fragment pGEX-hpc expression vector of recombinating, at the expression in escherichia coli fusion rotein; But its expression product is to exist with the inclusion body form; Need could obtain bioactive hepcidin through renaturation process, thereby utilize this type of pGEX-hpc expression vector to prepare the technology relative complex of hepcidin, cost is higher.([4] B.Srinivasulu, R.Syvitski, J.-K.Seo such as B.Srinivasulu; N.R.Mattatall; L.C.Knickle, S.E.Douglas.Expression, purificationand structural characterization of recombinant hepcidin; Anantimicrobial peptide identified inJapanese flounder; Paralichthys olivaceus [J] .Protein.Expr.Purif., 2008 (61): 36-44) report utilizes intestinal bacteria amalgamation and expression 6His-hepcidin, also mainly is that inclusion body is expressed.The applicant has proved also that in previous work large yellow croaker PC-hepc and black porgy AS-hepc2 mature peptide all have significant anti-microbial activity and (see [5] Ke-Jian Wang; Jing-JingCai; Ling Cai; Hai-Dong Qu; Ming Yang, Min Zhang.Cloning and expression of a hepcidin genefrom a marine fish (Pseudosciaena crocea) and the antimicrobial activity of its syntheticpeptide [J] .Peptides, 2009 (30): 638-646).

Summary of the invention

The object of the present invention is to provide tandem expression product and the structure of two kinds of fish Hepcidin tandem expression carriers and the preparation method of tandem expression product of two kinds of fish antibacterial peptide genes.

Technical scheme of the present invention is through making up the pET32/hepcidins expression vector of a kind of series connection large yellow croaker hepcidin (the cDNA sequence of leading peptide and mature peptide) and black porgy hepcidin (the cDNA sequence of leading peptide and mature peptide) gene; Transform host bacterium BL21 (DE3) abduction delivering soluble product; And purifying is with the method for the series connection hepcidin polypeptide of any label, thereby obtains the antimicrobial spectrum hepcidin tandem expression product wider than wherein a kind of hepcidin of single expression.

The series antimicrobial peptide hepcidins gene of the tandem expression product of said two kinds of fish antibacterial peptide genes comes from large yellow croaker antibacterial peptide hepcidin (being abbreviated as PC-hepc) and porgy antibiotic peptide hepcidin2 (being abbreviated as AS-hepc2) respectively; All comprise the leading peptide gene, both link to each other through the connection peptides encoding sox.

The structure of described two kinds of fish Hepcidin tandem expression carriers and the preparation method of tandem expression product are following:

1) structure of recombinant expression vector pET32/hepcidins

(1) synthesizes F1, R1, F2, four primers of R2 according to large yellow croaker antibacterial peptide PC-hepc gene and porgy antibiotic peptide AS-hepc2 gene; Wherein primers F 1 and primer R1 pairing amplification large yellow croaker hepcidin gene; Primers F 2 and primer R2 pairing amplification black porgy hepcidin gene; Primers F 1 and primer R2 pairing, the purpose product of connecting with hepcidins that the corresponding template reaction can be increased; After 35 PCR reactions, obtain large yellow croaker hepcidin gene, porgy hepcidin gene and hepcidin tandem gene respectively, agarose gel electrophoresis shows that respectively size is: 212bp, 222bp and 416bp;

(2) the hepcidins PCR product and the pET32a carrier that reclaim are carried out double digestion with NcoI and XhoI; Obtain having and preparatory dna fragmentation and the pET32a linear carrier of handling well of being connected; Through the gene clone ordinary method connect, conversion and mono-clonal screening and identify that enzyme is cut with sequencing result and shown that the hepcidins tandem sequence successfully is inserted into expression vector and reads sign indicating number correct;

2) abduction delivering of pET32/hepcidin recombinant plasmid in intestinal bacteria

With the thermal shock method pET32a empty plasmid and pET32/hepcidins recombinant plasmid are converted into abduction delivering in BL21 (DE3) competent cell respectively; The BL21 (DE3) that transforms with the pET32a empty plasmid is contrast, adopts polyacrylamide gel electrophoresis (SDS-PAGE) to detect fusion protein expression;

3) purifying of pET32/hepcidins recombinant plasmid fusion expressed product

(1) soluble analysis of hepcidins fusion expressed product

With the centrifugal collection thalline of the expression product of pET32/hepcidins recombinant plasmid in intestinal bacteria, with the PBS thalline that suspends, ultrasonication; Centrifugal ultrasonic liquid is got supernatant and is carried out the SDS-PAGE electrophoresis, confirms the solubility of expression product;

(2) affinity chromatography purifying hepcidin fusion expressed product

Collect thalline after will having a large amount of abduction deliverings of bacterial strain of higher solubility expression hepcidin; Adopt the metal chelate affinity chromatography post to carry out affinity chromatography; The metal chelate affinity chromatography filler is sepharose fast flow (GE Healthcare), and used chromatography reagent is solution A, B, C and D;

Said solution A is Ni ²⁺Metals ion, the Ni that the solution B flush away is unnecessary ²⁺, solution C balance chromatography column will be through the ultrasonic supernatant upper prop of expression bacterium behind the 0.45 μ m membrane filtration; Solution C is crossed the unconjugated albumen of post flush away; Solution D is crossed post wash-out target protein, collects elution peak, identifies through the SDS-PAGE electrophoresis to be the hepcidins fusion expressed product;

(3) endonuclease reaction of fusion rotein and the not purifying of tape label hepcidins polypeptide

With fusion rotein low temperature dialysis in the Tris-Cl damping fluid; Fully cut according to being with 6 * His recombinant enterokinase operational manual that this fusion rotein is carried out enzyme the dialysis back; Because can obtain the not hepcidins polypeptide of tape label sequence and fusion rotein after fusion protease is cut; And not fully cutting and carrier proteins and recombinant enterokinase 6 * His label is all arranged, enzyme cut the back mixture with enzyme cutting buffering liquid suitably dilute the back cross Ni with the 1.5ml/min flow velocity ²⁺Affinity column after the peak is passed in collection, just obtains desired polypeptides; The Hepcidins polypeptide is slowly changed dialyzate to ultrapure water from buffer A, removes salt constituents in the polypeptide.With-80 ℃ of frozen desired polypeptides that concentrate to frozen drying of cryogenic refrigerator after the 0.22 μ m membrane filtration degerming, the sample after lyophilize is handled places-20 ℃ of refrigerators to preserve again; Said buffer A is 50mM Tris-Cl, 50mM NaCl, and pH 8.0;

(4) mensuration of Hepcidins tandem polypeptide anti-microbial activity

A. microtitre meat soup dilution process: estimate the anti-microbial activity of hepcidins tandem polypeptide through measuring minimal inhibitory concentration;

B. with 10 times of MIC concentration doses of minimal inhibitory concentration hepcidins polypeptide streptococcus aureus is killed analysis; Set regular time and from culture hole, draw the volume that equates at interval; Carry out being inoculated into the plain agar plate after the same dilution, count every plate clone next day.

In (3) part of step 3), said Tris-Cl damping fluid is 20mM Tris-Cl, 150mM NaCl, and pH 8.0.

Through with after Trx TrxA merges the hepcidin gene solubility expression product that obtains connecting; Can pass through Ni affinity chromatography column purification; Polypeptide is cutting through the active enteropeptidase with 6 * His label behind the purifying; Enzyme is cut mixture again through Ni affinity chromatography column purification, collects two kinds of hepcidin series connection products that pass the peak and obtain not to be with any label, and gained hepcidin series connection product is used to tried the bacterial classification bacteriostatic activity and measures.

Porgy antibiotic peptide AS-hepc2 expression carrier pET32a of the present invention contains T7 promotor, Trx fusion rotein encoding sox and histidine-tagged (His-Tag); Utilize the tandem polypeptide gene of two restriction enzyme site clones of its NcoI and XhoI large yellow croaker PC-hepc gene order-linker-black porgy AS-hepc2 gene order, and obtain the pET32/hepcidins expression vector.Through the optimization expression condition; Solubility fused in tandem expression product can pass through N-terminal His-Tag affinitive layer purification; The fusion product of purifying can use the enteropeptidase enzyme that the Trx label in the fusion rotein is excised; Cross purification column once more, collect and pass the hepcidin fused in tandem expression product that the peak can obtain purifying.Learn that through calculating the albumen of pET32a empty carrier abduction delivering is 158aa, theoretical molecular is 17.1kDa; The albumen of Trx/hepcidins recombinant vectors abduction delivering is 292aa, and theoretical molecular is 32.2kDa.

The present invention is characterized in the contrast of existing hepcidin research report: the present invention is through hydrophobicity connection peptides (linker) connect large yellow croaker PC-hepc and black porgy AS-hepc2 gene; And merged the sulphur oxygen albumen also on the pET32a prokaryotic expression carrier; Induce down at the IPTG of low temperature and proper concn, give expression to the antibacterial peptide hepcidin soluble fusion protein of two kinds of fish in large quantities.Through affinitive layer purification this fusion rotein, utilize the active enteropeptidase cleavage of fusion proteins carry 6 * His again, through secondary affinity chromatography column purification, obtain having no the series connection hepcidin polypeptide of label.Bacteriostatic experiment shows that this series connection hepcidin polypeptide reaches 6 μ M and 6 μ M to gold-coloured staphylococci and staphylococcus epidermidis (gram-positive microorganism) MIC concentration, also has significant fungistatic effect to Aeromonas hydrophila, micrococcus lysodeikticus.In addition, the present invention selects leading peptide and the mature peptide sequence tandem expression with two hepcidin, and the result proves that the leading peptide of hepcidin and mature peptide tandem expression product also have significant anti-microbial activity.

Description of drawings

Fig. 1 is the amplification of hepcidin and hepcidins.In Fig. 1, (a) M:DNA Marker, DL2000; The 1:hepcidinsPCR product; 2: black porgy hepcidin; 3: large yellow croaker hepcidin; (b) M:DNA Marker, DL2000; The 4:pET32/hepcidins plasmid enzyme restriction is identified (NcoI and XhoI); (c) M:DNAMarker, DL2000; 5～9: expression strain positive colony bacterium liquid PCR identifies; 10: black porgy hepcidin; The arrow points band is the hepcidins tandem gene among the figure.

Fig. 2 is pET32a and pET32/hepcidins expression product SDS-PAGE electrophoresis.In Fig. 2, M:proteinmarker (KDa), Fermentas SM0431; 1:pET32a expresses the ultrasonic supernatant of bacterium; 2:pET32/hepcidins expresses the ultrasonic supernatant of bacterium.

Fig. 3 is a hepcidins fusion rotein SDS-PAGE electrophoresis before and after the purifying.In Fig. 3, M:protein marker FermentasSM0431; Sample behind the 1:hepcidins fusion rotein purifying; 2:pET32/hepcidins expresses the supernatant sample; A wears part for stream, and B is an elution peak.

Fig. 4 is a hepcidins fusion rotein enteropeptidase cutting effect.In Fig. 4, the 1:hepcidins fusion rotein; 2～6: enzyme is cut action time (down with), and 1,2,3,4,5h; M: albumen marker.

Fig. 5 cuts product for the hepcidins fusion protease and secondary is crossed affinity column SDS-PAGE electrophorogram.In Fig. 5, M:protein marker; The 1:hepcidins fusion protease is cut product and secondary and is crossed affinity column and pass; The 2:hepcidins fusion protease is cut product.

Fig. 6 kills the gold-coloured staphylococci curve mutually when growing for the hepcidins tandem polypeptide.In Fig. 6, X-coordinate is action time (min), and ordinate zou is that each time point (Tn) plate clone number and starting point (are 0min, T0) clone the ratio C FU (100%) of number.

Embodiment

Through embodiment the present invention is described further below.

1. the structure of recombinant expression vector pET32/hepcidins

1) contain the acquisition of the reorganization pPMD18-T positive plasmid template of series connection large yellow croaker hepcidin (the cDNA sequence of leading peptide and mature peptide) and black porgy hepcidin (the cDNA sequence of leading peptide and mature peptide) gene:

(1) utilizes Trizol test kit (available from Invitrogen company) to extract to carry out the RT-PCR amplification behind large yellow croaker, the black porgy juvenile fish liver total rna and carrying out the TA clone by ordinary method; And order-checking is correct; Plasmid places-20 ℃ of preservations, and it is frozen in-80 ℃ that bacterial classification adds 15% glycerine.

(2) with above-mentioned F1, R1, F2 and four primers of R2 of designing respectively, according to large yellow croaker antibacterial peptide PC-hepc gene (the Genbank accession number: EU443735) with porgy antibiotic peptide AS-hepc2 gene (Genbank accession number: AY669377) synthetic following primer:

F1:5 '-CAC CCATGG GTCCCAGCCAATGAAGAGCAAG-3 ' (underscore partly is the NcoI restriction enzyme site)

R1:5 '-

3 ' (bolded section is coding linker gene, and italics partly is amplification large yellow croaker hepcidin downstream primer)

F2:5 '-

3 ' (bolded section is coding linker gene, and italicized item is the upstream primer of amplification black porgy hepcidin gene)

R2:5 '-CA GAACCTGCAGCAGACACCAC-3 ' (underscore partly is the XhoI restriction enzyme site)

Being amplification template (the applicant makes up voluntarily and checks order correct) to comprise large yellow croaker, black porgy hepcidin (cDNA) reorganization pMD18-T plasmid respectively, is pairing primer amplification large yellow croaker hepcidin gene (comprising leading peptide and mature peptide encoding sox) with F1-R1, and amplification condition is: 100 μ l systems; 94 ℃, 5min; 94 ℃, 30s, 60 ℃, 30s, 72 ℃, 30s; Totally 35 circulations, pfu high-fidelity Taq enzymatic amplification (down together); F2, R2 are the upstream and downstream primer, amplification black porgy hepcidin gene (comprising leading peptide and mature peptide encoding sox), and reaction conditions is: 100 μ l systems, 94 ℃, 5min, 94 ℃, 30s, 60 ℃, 30s, 72 ℃, 30s, 35 circulations.Respectively go 5 μ l as the bridging template after utilizing sepharose to reclaim two kinds of hepcidin genes respectively, and be upstream and downstream primer amplification hepcidins (hepcidin-linker-hepcidin) tandem sequences with F1, R2.The pcr amplification condition is following: 100 μ l systems, and 94 ℃, 5min, 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 30s, 5 circulations, only changing annealing temperature is to react 30 circulations after 65 ℃ again.Three step pcr amplification purposes segmental reclaimer operations are following: pcr amplification product with 1.5% (W/V) sepharose-TAE electrophoretic separation after; Cutting-out contains the segmental sepharose of purpose, reclaims dna segment according to the operation of Qiaquick Gel Extraction Kit (available from QIAGEN) specification sheets.Because large yellow croaker hepcidin and black porgy hepcidin leading peptide and mature peptide have surpass 62% homology, and both downstream primer sequences only differ two bases, so be easy to mutual pairing; When using F1, R2 pairing primer; Just can amplify two DNA banding patterns, less is large yellow croaker hepcidin sequence, and bigger is the hepcidins tandem sequence; When reclaiming, only downcut bigger hepcidins sequence.Purpose fusion gene corresponding amino acid sequence is following among the expression vector pET32/hepcidins, and its destination gene expression product comprises enteropeptidase and cuts site, 6 * His-Tag and connection peptides aminoacid sequence (with the tandem polypeptide sequence called after hepcidins of its coding):

(3) hepcidins purpose fragment is cut, is connected and identify with the enzyme of pET32a carrier: with NcoI and two digestion with restriction enzyme hepcidins purpose fragments of XhoI and pET32a carrier, place 37 ℃ of incubators, more than the 8h; Utilize agarose gel electrophoresis to reclaim enzyme and cut back hepcidins fragment and pET32a linear carrier, the fragment after the recovery is carried out agarose gel electrophoresis again and is detected, and prepares 10 μ l linked systems according to yield; Adopt T4 ligase enzyme (available from precious biotechnology (Dalian) ltd), 16 ℃ of cool circulation waters are bathed and are spent the night, and next day is according to ordinary method transformed competence colibacillus DH5a; 10-20 positive colony of inferior daily aseptic toothpick picking serves as the pairing primer with F1 and R2, and bacterium colony PCR identifies clone's; Choose in two PCR positive colony inoculation 6ml LB liquid nutrient mediums 37 ℃ of 200rpm shaking table overnight cultures, alkaline lysis method of extracting plasmid DNA again; With NcoI and the two digestion with restriction enzyme recombinant plasmids of XhoI; Reaction system is plasmid 10 μ l, 10 * enzyme reaction buffer solution, 2 μ l, each 1 μ l of restriction enzyme; Aseptic MiliQ water is mended to total reaction volume 20 μ l, in 37 ℃ of incubator reaction 3h; Get 8 μ l reaction solutions and carry out 2% (W/V) sepharose-TAE electrophoresis evaluation.Can send company to carry out dna nucleotide sequence mensuration by digested recombinant clone.

Synthesize F1, R1, F2, four primers of R2 according to large yellow croaker antibacterial peptide PC-hepc gene and porgy antibiotic peptide AS-hepc2 gene; Wherein primers F 1 and primer R1 pairing amplification large yellow croaker hepcidin gene; Primers F 2 and primer R2 pairing amplification black porgy hepcidin gene; Primers F 1 and primer R2 pairing, the purpose product of connecting with hepcidins that the corresponding template reaction can be increased; After 35 PCR reactions, obtain large yellow croaker hepcidin gene, porgy hepcidin gene and hepcidin tandem gene respectively, agarose gel electrophoresis shows that respectively size is: 212bp, 222bp and 416bp (referring to Fig. 1).

2.pET32/hepcidins the abduction delivering of recombinant plasmid in intestinal bacteria

1) a small amount of abduction delivering: extract test kit in a small amount with plasmid and extract through the correct pET32/hepcidins recombinant plasmid of order-checking evaluation; With the thermal shock method pET32a empty plasmid and pET32/hepcidins recombinant plasmid are converted into respectively in BL21 (DE3) competent cell; Coat and express with LB agar plate (containing 50 μ g/ml Amp), 37 ℃ of overnight cultures; Single bacterium colony of the dull and stereotyped growth of some pET32a empty plasmids of picking and pET32/hepcidins recombinant plasmid transformed; Being inoculated in 10ml LB respectively expresses with (containing 50 μ g/ml Amp) in the substratum; 200rpm/min, 37 ℃ are cultured to bacteria concentration OD600 and are about 0.5～0.6, add then IPTG to final concentration be 0.4mMol/ml; At 29 ℃, 180rpm, shaking table is cultivated 7h.Centrifugal collection thalline, with the about 20ml suspension of PBS thalline, after the ultrasonication, 12000rpm, the centrifugal ultrasonic liquid of 30min is got supernatant, carries out the SDS-PAGE electrophoresis with 12% separation gel, the expression product (referring to Fig. 2) of solubility in the visible thalline supernatant.

3. affinity chromatography purifying hepcidin fusion expressed product

1) preparation of upper prop sample

The bacterial strain that will have higher solubility expression hepcidin, a large amount of 1 liter of LB substratum of abduction delivering, centrifugal collection thalline; With the PBS of precooling (15ml/g wet bacterium) suspension thalline, ice bath is placed 30min; Ultrasonication thalline to bacterium liquid is more inviscid under the ice bath; 12,000rpm high speed frozen centrifugation 30min, get supernatant with 0.45 μ m membrane filtration after, upper prop sample all set so far.

2) prepare the metal a flat iron plate for making cakes and close chromatography column

The metal chelate affinity chromatography filler is sepharose fast flow (GE Healthcare), behind the affinity chromatography medium dress post, crosses 5ml solution A a flat iron plate for making cakes earlier and closes the Ni2+ metals ion, with 3 column volume MiliQ washing posts; After the unnecessary Ni2+ of the solution B flush away of 3 column volumes, and with 3 column volume MiliQ washing posts; Followed 5 column volume solution C balance chromatography columns, appearance in the wait.

Chromatography reagent is:

Solution A: 200mM single nickel salt;

Solution B: 25mM NaH2PO4+500mM NaCl pH 4.0 (transferring pH) with phosphoric acid;

Solution C: 50mM phosphoric acid buffer+200mM NaCl+40mM imidazoles pH 8.0;

Solution D: 50mM phosphoric acid buffer+150mM NaCl+400mM imidazoles pH 8.0;

Complete soln is with 0.45 μ m membrane filtration.

3) go up column purification

With the whole upper props of solubility supernatant after filtering, cross post with the solution C of 10 column volumes simultaneously, the unconjugated albumen of flush away; Cross post wash-out target protein with the solution D of 5 column volumes again; Collect elution peak, take a morsel and carry out the evaluation of SDS-PAGE electrophoresis, calculate through gel scanning and learn that the fusion expressed product that scanning is coagulated behind the purifying has the purity (referring to Fig. 3) more than 90%.

4.Hepcidin the proteic endonuclease reaction of fused in tandem

With fusion rotein in Tris-Cl damping fluid (20mM Tris-Cl; 150mM NaCl (pH 8.0)) low temperature dialysis in, fully (referring to Fig. 4) cut according to being with 6 * His recombinant enterokinase (mcroorganism Science and Technology Ltd. in the Guangzhou) operational manual that this fusion rotein is carried out enzyme in the dialysis back.Because can obtain the not hepcidins polypeptide (hepcidins) of tape label sequence and fusion rotein after fusion protease is cut; And not fully cutting and carrier proteins and recombinant enterokinase 6 * His label is all arranged, enzyme cut the back mixture with enzyme cutting buffering liquid suitably dilute the back cross Ni with the 1.5ml/min flow velocity ²⁺Affinity column after the peak is passed in collection, just obtains desired polypeptides (referring to Fig. 5).The Hepcidins polypeptide is slowly changed dialyzate to ultrapure water from damping fluid (50mM Tris-Cl, 50mM NaCl pH 8.0), removes salt constituents in the polypeptide.Again with-80 ℃ of frozen desired polypeptides that concentrate to frozen drying of cryogenic refrigerator after the 0.22 μ m membrane filtration degerming.Sample after lyophilize is handled places-20 ℃ of refrigerators to preserve.

5.Hepcidin the mensuration of series protein anti-microbial activity

1) Application of B radford method is measured the BSA standard substance and is obtained the protein concentration typical curve.Can calculate the proteic concentration of hepcidins amalgamation and expression according to formula.

2) mensuration of Hepcidins tandem polypeptide anti-microbial activity

(1) estimates the anti-microbial activity of hepcidins tandem polypeptide through measuring minimal inhibitory concentration.

1. the dilution of protein sample

The dilution of target protein is carried out according to MIC setting concentration, uses μ M to dilute proportioning as unit, is that gradient is carried out on 96 hole microtest plates like 96,48,24,12,6,3,1.5 μ M.Protein sample will dilute proportioning again through 0.22 μ m filtering membrane filtration sterilization bacterium, and each concentration is provided with a parallel hole.

2. get tested bacterium, on the MH flat board, rule, be inverted and cultivate 12～16h; The picking mono-clonal is inoculated in the MH inclined-plane, continues overnight cultures and reaches the logarithmic growth stage in mid-term; Clean slant culture with DPBS, adjustment is diluted to OD600=0.1, and the dilution bacterium adding final volume of getting 18 μ l OD600=0.1 is that this bacteria concentration then is the MIC working concentration in the MH diluted medium (DPBS:600 μ l, MH:400 μ 1) of 1ml.Wherein staphylococcus epidermidis, streptococcus aureus culture temperature are 37 ℃, and Vibrio parahaemolyticus, micrococcus lysodeikticus, subtilis and Aeromonas hydrophila culture temperature are 28 ℃.

(2) MIC carries out on 96 porocyte culture plates, and every kind of tested bacterium is according to following operation setting blank group, negative control group and testing sample experimental group, and every group is provided with 2 parallel appearance:

1. blank group: add 50 μ l protein samples and 50 μ l DPBS;

2. negative control group: add 50 μ l bacteria suspensions and 50 μ l DPBS;

3. sample experimental group: add 50 μ d each concentration protein sample to be measured and 50 μ l bacteria suspensions;

28 ℃ cultivate 24h after, judge the MIC of hepcidins tandem polypeptide to bacterium, criterion is less than for this concentration hole bacterial count or equals half the in the control wells (not having the antibacterial peptide composition).

The result shows; Series connection hepcidins polypeptide all has dose-dependent bacteriostatic activity to staphylococcus epidermidis, streptococcus aureus, micrococcus lysodeikticus, Aeromonas hydrophila, subtilis, Corynebacterium glutamicum, intestinal bacteria pattern bacterium and cured shape genus bacillus (all available from DSMZ of Institute of Microorganism, Academia Sinica (CGMCC)), and hepcidins tandem polypeptide anti-microbial activity is referring to table 1.

Table 1

Strain name	Bacterial classification source and numbering	MIC(μM)
			Staphylococcus epidermidis	CGMCC?1.2429	3～6
Streptococcus aureus	CGMCC?1.363	3～6
			Micrococcus lysodeikticus	CGMCC?1.643	6～12
Subtilis	CGMCC?1.108	12～24
			Corynebacterium glutamicum	CGMCC?1.1886	12～24
Aeromonas hydrophila	CGMCC?1.1801	6～12
			Intestinal bacteria pattern bacterium	CGMCC?1.2389	＞48
Cured shape genus bacillus	CGMCC?1.447	＞48

(3) with 10 times of MIC (60 μ M) concentration dose hepcidins polypeptide streptococcus aureus is killed analysis; Set regular time and from culture hole, draw the volume that equates at interval; Carry out being inoculated into the plain agar plate after the same suitable dilution; Count every plate clone next day, CFU (is 0min, T0) clones the ratio of number: CFU=Tn/T0*100% (n=15,30,60,90,120 and 180min) (referring to Fig. 6) for each time point (Tn) plate clone number and starting point in the graphic representation.

Claims (2)

1. the tandem expression product of two kinds of fish antibacterial peptide genes; The series antimicrobial peptide hepcidins gene that it is characterized in that the tandem expression product of said two kinds of fish antibacterial peptide genes comes from large yellow croaker antibacterial peptide hepcidin and porgy antibiotic peptide hepcidin2 respectively; All comprise the leading peptide gene; Both link to each other through the connection peptides encoding sox, and the encoding sequence of large yellow croaker antibacterial peptide hepcidin is:

gccatggcga?atggtgtccc?agccaatgaa?gagcaagagc?tggagcagca?aatttatttt 60

gctgatccag?agatgccagt?ggaatcatgc?aagatgccgt?attacatgcg?tgagaatcgt 120

cagggcagcc?ctgctagatg?caggttttgc?tgccgttgct?gtcctagaat?gaggggatgt 180

ggtatctgct?gcaggttc 198

The encoding sequence of porgy antibiotic peptide hepcidin2 is:

ggctcattca?ctgaggtgca?agagccggag?gagccaatga?acaatgagag?tccagttgct 60

gcacatgaag?agaagtcaga?ggagtcctgg?aagatgccgt?ataacaacag?acacaagcgc 120

agccccgctg?gttgtcgctt?ttgctgcggt?tgctgtccta?acatgagagg?atgtggtgtc 180

tgctgcaggt?tctaactcga?g 201

The encoding sequence of connection peptides is:

5’-ggttctccaggttccggt-3’；

Said large yellow croaker antibacterial peptide hepcidin gene and porgy antibiotic peptide hepcidin2 gene are respectively in the American National biotechnology Genbank of information center accession number: EU443735 and AY669377.

2. the preparation method of the tandem expression product of two kinds of fish antibacterial peptide genes as claimed in claim 1 is characterized in that may further comprise the steps:

1) structure of recombinant expression vector pET32/hepcidins

(1) according to large yellow croaker antibacterial peptide hepcidin gene and synthetic following 4 primers of porgy antibiotic peptide hepcidin2 gene:

F1：5′-CACCCATGGCGGTCCCAGCCAATGAAGAGCAAG-3′

R1：5′-ACCGGAACCTGGAGAACCGAACCTGCAGCAGATACC-3′

F2：5′-GGTTCTCCAGGTTCCGGTGGCTCATTCACTGAGGTGC-3′

R2：5′-CAGCTCGAGTTAGAACCTGCAGCAGACACCAC-3′

F1, R1 pairing amplification large yellow croaker hepcidin gene wherein, F2, R2 pairing amplification black porgy hepcidin2 gene, F1, R2 pairing, the purpose product of connecting with hepcidins that the corresponding template reaction can be increased; After 35 PCR reactions, obtain large yellow croaker hepcidin gene, black porgy hepcidin2 gene and hepcidins tandem gene respectively, agarose gel electrophoresis shows that respectively size is: 212bp, 222bp and 416bp;

(2) the hepcidins PCR product and the pET32a carrier that reclaim are carried out double digestion with NcoI and XhoI; Obtain having and preparatory dna fragmentation and the pET32a linear carrier of handling well of being connected; Through the gene clone ordinary method connect, conversion and mono-clonal screening and identify that enzyme is cut with sequencing result and shown that the hepcidins tandem sequence successfully is inserted into expression vector and reads sign indicating number correct;

2) abduction delivering of pET32/hepcidin recombinant plasmid in intestinal bacteria

With the thermal shock method pET32a empty plasmid and pET32/hepcidins recombinant plasmid are converted into abduction delivering in BL21 (DE3) competent cell respectively, the BL21 (DE3) that transforms with the pET32a empty plasmid is contrast, adopts SDS-PAGE electrophoresis detection fusion protein expression;

3) purifying of pET32/hepcidins recombinant plasmid fusion expressed product

(1) soluble analysis of hepcidins fusion expressed product

With the centrifugal collection thalline of the expression product of pET32/hepcidins recombinant plasmid in intestinal bacteria, with the PBS thalline that suspends, ultrasonication; Centrifugal ultrasonic liquid is got supernatant and is carried out the SDS-PAGE electrophoresis, confirms the solubility of expression product;

(2) affinity chromatography purifying hepcidins tandem gene

Collect thalline after will having a large amount of abduction deliverings of bacterial strain of higher solubility expression hepcidin, adopt the metal chelate affinity chromatography post to carry out affinity chromatography, the metal chelate affinity chromatography filler is chelating sepharose fast flow, and used chromatography reagent is:

Solution A: 200mM single nickel salt;

Solution B: 25mM NaH ₂PO ₄+ 500mM NaCl pH 4.0;

Solution C: 50mM phosphoric acid buffer+200mM NaCl+40mM imidazoles pH 8.0;

Solution D: 50mM phosphoric acid buffer+150mM NaCl+400mM imidazoles pH 8.0;

Said solution A is Ni ²⁺Metals ion, the Ni that the solution B flush away is unnecessary ²⁺, solution C balance chromatography column will be through the ultrasonic supernatant upper prop of expression bacterium behind the 0.45 μ m membrane filtration; Solution C is crossed the unconjugated albumen of post flush away; Solution D is crossed post wash-out target protein, collects elution peak, identifies through the SDS-PAGE electrophoresis to be the hepcidins fusion expressed product;

(3) endonuclease reaction of fusion rotein and the not purifying of tape label hepcidins polypeptide

With fusion rotein in 20mM Tris-Cl; 150mM NaCl; Low temperature dialysis in the pH 8.0 Tris-Cl damping fluids, fully cut according to being with 6 * His recombinant enterokinase operational manual that this fusion rotein is carried out enzyme the dialysis back, because fusion protease can obtain the not hepcidins polypeptide of tape label sequence and fusion rotein after cutting; And not fully cutting and carrier proteins and recombinant enterokinase 6 * His label is all arranged, enzyme cut the back mixture with enzyme cutting buffering liquid suitably dilute the back cross Ni with the 1.5ml/min flow velocity ²⁺Affinity column after the peak is passed in collection, just obtains desired polypeptides; The Hepcidins polypeptide is slowly changed dialyzate to ultrapure water from damping fluid E, removes salt constituents in the polypeptide; With-80 ℃ of frozen desired polypeptides that concentrate to frozen drying of cryogenic refrigerator after the 0.22 μ m membrane filtration degerming, the sample after lyophilize is handled places-20 ℃ of refrigerators to preserve again; Said damping fluid E is 50mM Tris-Cl, 50mM NaCl pH8.0;

(4) mensuration of Hepcidins tandem polypeptide anti-microbial activity

A. microtitre meat soup dilution process: estimate the anti-microbial activity of hepcidins tandem polypeptide through measuring minimal inhibitory concentration;

B. with 10 times of MIC concentration doses of minimal inhibitory concentration hepcidins polypeptide streptococcus aureus is killed analysis; Set regular time and from culture hole, draw the volume that equates at interval; Carry out being inoculated into the plain agar plate after the same dilution, count every plate clone next day.

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