CN102517293A - Expression vector of Acanthopagrus schlegelii hepcidin and construction method, expression product and preparation method thereof - Google Patents
Expression vector of Acanthopagrus schlegelii hepcidin and construction method, expression product and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an expression vector of Acanthopagrus schlegelii hepcidin and a construction method, an expression product and a preparation method thereof, which relate to fish genetic engineering. According to a complementary deoxyribonucleic acid (cDNA) sequence of the obtained Acanthopagrus schlegelii hepcidin (AS-hepc2), induced expression is performed on an AS-hepc2 precursor peptide fusion protein in escherichia coli by using a pET28a+prokaryotic expression vector to obtain a soluble expression product; affinity chromatography purification is performed on the soluble expression product to obtain a target protein used for measuring antibacterial activity. A pure protein can be obtained only by performing affinity purification for one time without performing P3C protease cutting or tagging on a constructed BL21(DE3) (pET28a/AS-hepc2) expression product, thereby, the operating steps are simplified; the operating cost is reduced; and the soluble pure protein is rapidly obtained. The induced expression is performed on the constructed BL21(DE3) (pET28a/AS-hepc2) expression product for 5 hours, thereby, the amount of the pure protein obtained after nickel affinity purification is performed on the BL21(DE3) (pET28a/AS-hepc2) expression product can be 10-50 times the amount of the target protein and the soluble pure protein, and the expression efficiency is high.
Description
Technical field
The present invention relates to the fish genetically engineered in the biological technical field, especially relate to porgy antibiotic peptide (Hepcidin) expression vector and construction process and expression product and preparation method.
Background technology
Antibacterial peptide is the important composition composition of marine animal innate immune system.Research, exploitation and application antibacterial peptide; With effectively solving the bacterial drug resistance problem that faces in the sea farming production; And will improve because problems such as fishery products drug residue that abuse of antibiotics causes in the feed Ingredient and water environment pollution have important scientific meaning and actual application value.
Hepcidin is the cationic antibacterial peptide that halfcystine is rich in one type of conservative site of C end.The applicant (1.Ming Yang; Ke-Jian Wang; Jun-Hui Chen, Hai-Dong Qu, Shao-Jing Li.Genomic organization and tissue-specific expression analysis of hepcidin-like genes from black porgy [J] Fish Shellfish Immun; 2007; 23:1060-1071) obtained 7 hepcidin-like genes (AS-hepc 1～7) in report clone from China breed black porgy in 2007, the expression amount of porgy antibiotic peptide hepcidin mRNA obviously rises under bacteria artificial infects, and has explained that this antibacterial peptide has important immunization in the black porgy body.And confirmed that on protein level the AS-hepc2 of synthetic and AS-hepc6 mature peptide all have lethal effect (2.Ming Yang to gram-positive microorganism and Gram-negative bacteria; Bei Chen; Jing-Jing Cai; Hui Peng; Ling-Cai, Jian-Jun Yuan, Ke-Jian Wang.Molecular characterization of hepcidin AS-hepc2 and AS-hepc6 in black porgy (Acanthopagrus schlegelii): Expression pattern responded to bacterial challenge and in vitro antimicrobial activity [J] Comp Biochem Physiol BBiochem Mol Biol.; 2011,158 (2): 155-163).
Black porgy AS-hepc2 is liver specific expression's a hepcidin variant, and its gene (GenBank accession numbers:AY669377) is by 267 based compositions, 88 amino acid of encoding
[1]The same with other hepcidin antibacterial peptide structures that have been found that, AS-hepc2 is by the signal peptide (preceding 24 amino acid) of N end, leading peptide (40 amino acid) and mature peptide (24 the amino acid) composition that is positioned at the C end.The AS-hepc2 precursor peptide once by escherichia coli prokaryotic expression system Top10F ' (pTrc-CKS/hepcidin) (3. Wang Ke is hard, Yang Ming, Cai Jingjing. the expression vector of black porgy antibiotic peptide Hepcidin and expression product and structure preparation method [P] thereof. Chinese patent: 200710008862; 2007-4-20) and pichia spp eukaryotic expression system GS115 (pIC9K/AS-hepc2) (4. Cai's Jingjing; Yang Ming, Cai Ling, Guo Qing; Pan Ruizhen; Wang Kejian. expression and the anti-microbial activity [J] thereof of porgy antibiotic peptide hepcidin in pichia spp. Xiamen University's journal (natural science edition), 2009,48 (5): 738-743) obtain the vivoexpression product.But owing to obtaining reasons such as pure article complex procedures of albumen or technology be limited, caused the recombinant antibacterial peptide hepcidin yield that obtains not high, perhaps antimicrobial spectrum research is insufficient.Therefore, need to set up AS-hepc2 expression vector more efficiently, thereby obtain the great expression product.
Summary of the invention
The object of the present invention is to provide a kind of porgy antibiotic peptide expression vector and construction process and expression product and preparation method.
The gene order of porgy antibiotic peptide AS-hepc2 is:
ggctcattca ctgaggtgca agagccggag gagccaatga acaatgagag tccagttgct 60
gcacatgaag agaagtcaga ggagtcctgg aagatgccgt ataacaacag acacaagcgc 120
agccccgctg gttgtcgctt ttgctgcggt tgctgtccta acatgagagg atgtggtgtc 180
tgctgcaggttc 192
The aminoacid sequence of porgy antibiotic peptide AS-hepc2 is:
Gly Ser Phe Thr Glu Val Gln Glu Pro Glu Glu Pro Met Asn Asn Glu
1 5 10 15
Ser Pro Val Ala Ala His Glu Glu Lys Ser Glu Glu Ser Trp Lys Met
20 25 30
Pro Tyr Asn Asn Arg His Lys Arg Ser Pro Ala Gly Cys Arg Phe Cys
35 40 45
Cys Gly Cys Cys Pro Asn Met Arg Gly Cys Gly Val Cys Cys Arg Phe
50 55 60
The preparation method of said porgy antibiotic peptide AS-hepc2 may further comprise the steps:
1) recombinant expression vector of structure porgy antibiotic peptide AS-hepc2;
2) step 1) gained recombinant expression vector is imported host cell, and host cell is carried out abduction delivering, obtain expression product;
3) purification procedures 2) expression product of gained, obtain recombinant protein, i.e. porgy antibiotic peptide AS-hepc2 pro protein.
In step 1), said expression vector is selected pET28a+ for use.
In step 2) in, said host cell can be intestinal bacteria E.coli BL21 (DE3) pLys etc.
In step 3), the concrete grammar of said separation and purification is: thalline behind the abduction delivering is carried out ultrasonication, and supernatant after the centrifugal collection ultrasonication carries out affinitive layer purification.
The present invention is according to the porgy antibiotic peptide AS-hepc2 cDNA sequence that obtains; Utilize the pET28a+ prokaryotic expression carrier; Abduction delivering AS-hepc2 precursor peptide fusion rotein in intestinal bacteria obtains the solubility expression product, through the target protein that obtains behind the affinitive layer purification; In order to measure anti-microbial activity, lay the foundation for studying black porgy hepcidin function and application.
The escherichia coli prokaryotic expression system Top10F ' that in the past made up (pTrc-CKS/hepcidin) need through P3C proteolytic enzyme cutting CKS label and secondarily purified after could obtain the pure article of hepcidin albumen.The BL21 (DE3) that the present invention makes up (pET28a/AS-hepc2) expression product need not through P3C proteolytic enzyme cutting label; Only need to obtain the pure article of albumen through an affinity purification; Simplify operation steps greatly, reduced running cost, obtained the pure article albumen of solubility more apace.Simultaneously, (pTrc-CKS/hepcidin) owing to need through secondarily purified, loss of proteins is bigger for prokaryotic expression system Top10F ', and the final pure article protein content that obtains is lower.Pichia spp eukaryotic expression system GS115 (pIC9K/AS-hepc2) expressing quantity behind 120h behind the abduction delivering also is merely 1.1mg/L (4. Cai's Jingjing, Yang Ming, Cai Ling; Guo Qing; Pan Ruizhen, Wang Kejian. expression and anti-microbial activity [J] the Xiamen University journal (natural science edition) thereof of porgy antibiotic peptide hepcidin in pichia spp, 2009; 48 (5): 738-743); The BL21 (DE3) that the present invention makes up is expression system abduction delivering 5h again (pET28a/AS-hepc2), and the pure article amount of the albumen that behind nickel post affinity purification, obtains can be the two 10～50 times, has improved expression efficiency greatly.
The black porgy AS-hepc2 fusion rotein of said prokaryotic expression can selectivity suppresses the growth of gram-positive microorganism and negative bacterium; For example gram-positive microorganism streptococcus aureus (Staphylococcus aureus), micrococcus luteus (Micrococcus.luteus) and Gram-negative bacteria Pseudomonas fluorescens (Pseudomonas fluorescens), Pseudomonas stutzeri (Pseudomonas stutzer); The growth of shigella flexneri (Shigella flexneri) has potential using value in the exploitation of resisting pathogenic microbes new drug and animal feedstuff additive.
Description of drawings
Fig. 1 is pET28a+ expression vector restriction enzyme digestion and electrophoresis figure.M is DNA Marker DL2000, and 1 is that pET28a+ carrier enzyme is cut the back fragment.
Fig. 2 is an AS-hepc2 PCR electrophoretogram.M is DNA Marker DL2000, and 1 is AS-hepc2 PCR fragment.
Fig. 3 is a pET28a/AS-hepc2 prokaryotic expression carrier design of graphics.
Fig. 4 analyzes the abduction delivering situation of recombinant plasmid pET28a/AS-hepc2 in intestinal bacteria for SDS-PAGE.1 for pET28a/AS-hepc2 before IPTG induces, 2～7 for pET28a/AS-hepc2 after IPTG induces, M is protein MarkerSM0441 (a Fermentas company).
Fig. 5 analyzes abduction delivering product soluble analysis for SDS-PAGE.M is protein Marker SM0441 (a Fermentas company), and 1 for inducing thalline ultrasonication supernatant, and 2 for inducing thalline ultrasonication deposition.
Fig. 6 analyzes IMAC eluted protein electrophoretogram for SDS-PAGE.Protein Marker SM0431 (Fermentas company), 1 is that stream is worn albumen, and 2 is 10% solution B eluted protein, and 3 is 100% solution B eluted protein.
Embodiment
Below be described with reference to the accompanying drawings technical scheme of the present invention through embodiment.
The structure of embodiment 1 porgy antibiotic peptide AS-hepc2 recombinant expression vector
1) processing of pET28a+ carrier
Before clone's goal gene, from plasmid clone bacterial strain DH5 α (pET28a+), extract the pET28a plasmid in a small amount, concrete operation method is seen east victory plasmid extraction kit.
Get the pET28a+ plasmid of the above-mentioned purifying of 30 μ L, with Nco I/Xho I Restriction Enzyme double digestion, hatch 5h for 37 ℃, enzyme is cut system such as following table; After reaction finishes, win the enzyme reaction product purification kit with east and remove enzyme and Nucleotide fragment; 1% (W/V) sepharose-TAE electrophoresis detection enzyme is cut efficient; Reclaim test kit with the Qiagen gel and reclaim specific fragment, be stored in-20 ℃ for use.(referring to Fig. 1)
2) acquisition of porgy antibiotic peptide AS-hepc2 precursor peptide gene
According to the carrier MCS, the upstream and downstream primer of purpose of design gene:
Upstream primer 5 ' end adds Nco I restriction enzyme site;
28-prohep2f：5’CATG
CCATGG gc tca ttc act gag gt 3’
Downstream primer 5 ' end adds Xbo I restriction enzyme site;
28-prohep2r：5’CCG
CTCGAG gaa cct gca gca gac acc 3’
With black porgy AS-hepc2 (cDNA) reorganization pPMD18-T positive plasmid is amplification template, and with corresponding upstream and downstream primer separately, with pfu high-fidelity Taq enzymatic amplification purpose fragment, reaction system is following:
Mix, on thermal cycler, carry out the PCR reaction according to following program:
①94℃ 2min
2. 30 circulations:
94℃ 30sec
60℃ 30sec
72℃ 30min
③72℃ 10min
④16℃ Forever
Reaction is carried out 2% (W/V) sepharose-TAE electrophoresis with all reaction solutions after finishing, and reclaims test kit with the Qiagen gel and reclaims specific fragment.(referring to Fig. 2)
3) connect the preparation of dna fragmentation in advance
With Nco I/Xho I Restriction Enzyme double digestion, reaction solution places 37 ℃ of water-baths to react 5h with the AS-hepc2 product that reclaims.The enzyme system of cutting is specially:
Reaction is cut efficient with 2% (W/V) sepharose-TAE electrophoretic examinations enzyme after finishing; Reclaim test kit with the Qiagen gel again and reclaim specific fragment, the goal gene that obtains has and the mutual paired sticky end of handling well of linear carrier.
4) structure of expression vector, conversion and evaluation
To pass through the pET28a+ prokaryotic expression carrier that has identical sticky end after a series of processing and be connected with black porgy AS-hepc2, reaction system is following:
16 ℃ of incubated overnight; Next day, get 5 μ L ligation liquid and be converted in the 50 μ L E.coli DH5 α competent cells, be coated on overnight cultures on the LB agar plate that contains kantlex (50 μ g/mL).Next day, picking list bacterium colony was identified the positive colony bacterium with the PCR method, gave birth to worker company by Shanghai and carried out dna nucleotide sequence mensuration.
The result proves and connects correctly that the ORFs of Nucleotide (ORF) coding finally obtains the pET28a/AS-hepc2 fusion expression vector that correctly makes up continuously.The fusion rotein that this expression vector gives expression to will connect 6 * His taq at the carbon teminal of AS-hepc2, thereby pass through Ni
+But affinity chromatography purifying AS-hepc2 albumen.The structure iron of carrier is referring to Fig. 3.
The abduction delivering of embodiment 2pET28a/AS-hepc2 recombinant plasmid in e. coli bl21 (DE3)
Express with LB agar plate (50 μ g/mL Kan), 37 ℃ of overnight cultures identifying correct pET28a/AS-hepc2 recombinant plasmid transformed to E.coliBL21 (DE3) competent cell, coating;
At bacterium colony hour, the some single bacterium colonies of picking are inoculated in 20ml LB respectively and express with (50 μ g/mL Kan) in the substratum, 90～120rpm, and 25 ℃ of overnight cultures are to OD
600Be about 0.5, add then IPTG to final concentration be 100 μ g/mL, cultivate the 5h abduction deliverings at 30 ℃ of following 180rpm shaking tables.Utilize polyacrylamide gel electrophoresis (SDS-PAGE) to detect fusion protein expression.
The result shows that pET28a/AS-hepc2 has the obvious expression protein band before after inducing, inducing; Recombinant bacterial strain induces the molecular weight of back expressing protein about 19kDa.(referring to Fig. 4)
Embodiment 3pET28a/AS-hepc2 recombinant plasmid is at the soluble analysis of expression in escherichia coli product
By method conversion as stated, cultivation 5ml seed liquor, get 1mL and add to (50 μ g/mL Kan) among the 20mL LB, 37 ℃ of following 180～200rpm, shaking table is cultured to OD
600=0.5, add IPTG to concentration be 100 μ g/ml, induce 5h for 30 ℃; Centrifugal collection thalline is with the about 2ml suspension of PBS thalline, ultrasonication; 12000g, the centrifugal ultrasonic liquid of 15min is got cleer and peaceful deposition respectively, carries out the SDS-PAGE electrophoresis.
The result shows: the AS-hepc2 fusion expressed product exists with two kinds of forms of cleer and peaceful inclusion body on the solubility, but major part is present in the solvable supernatant.(referring to Fig. 5)
1) preparation of upper prop sample
A large amount of abduction delivering 3～4L, centrifugal collection thalline; With the PBS of precooling (15mL/g wet bacterium) suspension thalline, ice bath is placed 30min, stirs frequently; Ultrasonication thalline to bacterium liquid more inviscid (power 20%, 5 second 10 seconds at interval) under the ice bath; 12,000rpm high speed frozen centrifugation 20min.Supernatant is treated column purification after through 0.45 μ m membrane filtration.
2) the ultrasonic supernatant of affinity chromatography column purification
Chromatography reagent is following:
Solution A: 200mM single nickel salt
Solution B: 25mM NaH
2PO
4+ 500mM NaCl pH 4.0 (phosphoric acid is transferred pH)
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
Solution E: 50mM EDTA+300mM NaCl pH 8.0
Clean 20% ethanol on the chromatography column with 5～10 volumes MilliQ water, 3～5 column volume solution A combine Ni
+, 5～10 column volume solution B flush awaies are the Ni of column not
+, 5～10 volumes solution C balance HisTrap chromatography columns with the solubility supernatant upper prop after filtering, are crossed post with the solution C of 5～10 column volumes, the unconjugated albumen of flush away; Cross post with 10% solution D, 5～10 column volumes, flush away bonded nonspecific proteins; Cross post with 100% solution D, 5～10 column volumes, wash-out target protein; Collect elution peak, take a morsel and carry out the evaluation of SDS-PAGE electrophoresis.
The result shows; The rAS-hepc2 fusion expressed product can specificly combine with nickel ion chelating affinity column; After the imidazoles solution competition washing of higher concentration, fusion expressed product is by wash-out, and acquisition purity is 95.8% reorganization AS-hepc2 fusion expressed product.(referring to Fig. 6)
3) desalination of eluted protein
AS-hepc2 under the affinity chromatography wash-out is put into dialysis tubing, and dialyzate is repeatedly changed in dialysis in low salts solution (50mM Tris+50mM NaCl, pH 8.0) under 4 ℃.Subsequently under 4 ℃ in MilliQ water dialysed overnight, change MilliQ water 3 times.Sample takes out from dialysis tubing, deposit in-80 ℃ for use.
4) mensuration of recombinant protein minimal inhibitory concentration (MIC)
Protein sample through 0.22 μ m filtering membrane filtration sterilization after, Application of B radford method is measured the BSA standard substance and is obtained the protein concentration typical curve, can calculate the concentration of AS-hepc2 recombinant protein according to formula.With the protein solution doubling dilution to 3-96 μ M.
The mensuration of MIC (Minimum Inhibitory Concentration, minimal inhibitory concentration) is with reference to the method for Bulet etc.
[5,6], on 96 porocyte culture plates, carry out.
(1) gets streptococcus aureus, micrococcus lysodeikticus, micrococcus luteus, subtilis, wax shape bacillus, have a liking for steam Zymomonas mobilis, Pseudomonas fluorescens, Pseudomonas stutzeri, shigella flexneri, Pseudomonas aeruginosa and on the MH flat board, rule; The picking mono-clonal is inoculated in the inclined-plane, continues to cultivate 12～16h; 10mM sodium phosphate salt damping fluid (pH 7.2) flushing bacterium slant culture, adjustment is diluted to OD
600=0.03 is subsequent use.
(2) antibacterial experiment carries out on 96 porocyte culture plates with reference to the method for (1993) such as Bulet P., 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 3 parallel appearance:
1. positive controls: add 50 μ L sodium phosphate salt damping fluids and 30 μ L bacteria suspensions;
2. blank group: add 50 μ L testing protein samples and 30 μ L sodium phosphate salt damping fluids;
3. sample experimental group: add 50 μ L testing protein samples and 30 μ L bacteria suspensions;
(3) every hole adds 20 μ L MH liquid nutrient mediums, and optimum temperuture is cultivated 24-48h, observations.
MIC result to 14 kinds of bacteria-measurings shows; The black porgy AS-hepc2 fusion rotein of prokaryotic expression can selectivity suppresses the growth of gram-positive microorganism and negative bacterium; For example gram-positive microorganism micrococcus luteus (Micrococcus.luteus) streptococcus aureus (Staphylococcus aureus) and Gram-negative bacteria Pseudomonas fluorescens (Pseudomonas fluorescens), Pseudomonas stutzeri (Pseudomonas stutzer), the growth of shigella flexneri (Shigella flexneri).AS-hepc2 recombination expression product anti-microbial activity is referring to table 1.
Table 1AS-hepc2 recombination expression product anti-microbial activity
CGMCC no: Chinese common micro-organisms culture presevation administrative center numbering; CMCC, medical microbial culture presevation administrative center.
Claims (6)
1. the gene order of porgy antibiotic peptide AS-hepc2 is:
ggctcattca ctgaggtgca agagccggag gagccaatga acaatgagag tccagttgct 60
gcacatgaag agaagtcaga ggagtcctgg aagatgccgt ataacaacag acacaagcgc 120
agccccgctg gttgtcgctt ttgctgcggt tgctgtccta acatgagagg atgtggtgtc 180
tgctgcaggttc 192。
2. the aminoacid sequence of porgy antibiotic peptide AS-hepc2 is:
Gly Ser Phe Thr Glu Val Gln Glu Pro Glu Glu Pro Met Asn Asn Glu
1 5 10 15
Ser Pro Val Ala Ala His Glu Glu Lys Ser Glu Glu Ser Trp Lys Met
20 25 30
Pro Tyr Asn Asn Arg His Lys Arg Ser Pro Ala Gly Cys Arg Phe Cys
35 40 45
Cys Gly Cys Cys Pro Asn Met Arg Gly Cys Gly Val Cys Cys Arg Phe
50 55 60。
3. the preparation method of porgy antibiotic peptide AS-hepc2 is characterized in that may further comprise the steps:
1) recombinant expression vector of structure porgy antibiotic peptide AS-hepc2;
2) step 1) gained recombinant expression vector is imported host cell, and host cell is carried out abduction delivering, obtain expression product;
3) purification procedures 2) expression product of gained, obtain recombinant protein, i.e. porgy antibiotic peptide AS-hepc2 pro protein.
4. the preparation method of porgy antibiotic peptide AS-hepc2 as claimed in claim 3 is characterized in that in step 1) said expression vector is selected pET28a+ for use.
5. the preparation method of porgy antibiotic peptide AS-hepc2 as claimed in claim 3 is characterized in that in step 2) in, said host cell is intestinal bacteria E.coli BL21 (DE3) pLys.
6. the preparation method of porgy antibiotic peptide AS-hepc2 as claimed in claim 3; It is characterized in that in step 3); The concrete grammar of said separation and purification is: thalline behind the abduction delivering is carried out ultrasonication, and supernatant after the centrifugal collection ultrasonication carries out affinitive layer purification.
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| CN110776560A (en) * | 2019-10-09 | 2020-02-11 | 厦门大学 | Sparus latus antibacterial peptide AS-hepc3 (48-56)And uses thereof |
Citations (1)
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| CN101063145A (en) * | 2007-04-20 | 2007-10-31 | 厦门大学 | Expression carrier for black porgy antibiotic peptide Hepcidin and expression product and constructing preparation method |
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| CN101063145A (en) * | 2007-04-20 | 2007-10-31 | 厦门大学 | Expression carrier for black porgy antibiotic peptide Hepcidin and expression product and constructing preparation method |
Non-Patent Citations (2)
| Title |
|---|
| 杨明: "养殖真鲷、黑鲷抗菌肽hepcidin的基因克隆、表达特性及其抗菌活性研究", 《中国博士学位论文全文数据库（农业科技辑）》, no. 1, 15 July 2007 (2007-07-15), pages 052 - 2 * |
| 蔡晶晶: "黑鲷抗菌肽hepcidin 在毕赤酵母中的表达及其抗菌活性", 《厦门大学学报(自然科学版)》, vol. 48, no. 5, 30 September 2009 (2009-09-30), pages 2 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110776560A (en) * | 2019-10-09 | 2020-02-11 | 厦门大学 | Sparus latus antibacterial peptide AS-hepc3 (48-56)And uses thereof |
| US11299514B2 (en) | 2019-10-09 | 2022-04-12 | Xiamen University | Antimicrobial peptide AS-hepc3(48-56) of Acanthopagrus schlegelii and method thereof |
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Application publication date: 20120627 |