CN102433341B

CN102433341B - Prokaryotic expression product of epinephelus coioides antibacterial peptide and preparation method thereof

Info

Publication number: CN102433341B
Application number: CN201110386427.6A
Authority: CN
Inventors: 王克坚; 曲海东; 陈贝
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2011-11-28
Filing date: 2011-11-28
Publication date: 2014-02-26
Anticipated expiration: 2031-11-28
Also published as: CN102433341A

Abstract

The invention provides a prokaryotic expression product of epinephelus coioides antibacterial peptide and a preparation method thereof and relates to genetic engineering expression of fishes in the technical field of biology. A constructed expression vector containing an EC-hepcidin3 gene can be subjected to induced expression in escherichia coli; and a large amount of expression products are obtained in vitro by purifying the expression product at high efficiency. The expression vector containing the EC-hepcidin3 gene is a recombinant expression vector pET28a/EC-hepcidin 3 constructed by inserting an EC-hepcidin 3 precursor peptide gene sequence in a prokaryotic expression vector pET28a+. The expression product obtained in vitro is a recombinant protein EC-proHep3 obtained by induced expression and purification of the recombinant expression vector pET28a/EC-hepcidin 3 in the escherichia coli.

Description

Prokaryotic expression product of a kind of Epinephelus coioides antimicrobial petide and preparation method thereof

Technical field

The fish gene engineering the present invention relates in biological technical field is expressed, and especially relates to expression vector and expression product and the structure preparation method thereof of a kind of Epinephelus coioides antimicrobial petide Hepcidin3.

Background technology

Antibacterial peptide (Antimicrobial peptides, AMPs) be the important composition composition of fish innate immune system, fish can secrete and produce the invasion and attack that multiple antibacterial peptide is resisted pathogenic micro-organisms a large amount of in aquatic environment, and these pathogenic micro-organisms comprise various bacteria, fungi and virus etc.Utilize Protocols in Molecular Biology, obtain in vitro a large amount of, activated antibacterial peptide product, further these antibacterial peptide products of application may strengthen the immunizing power of aquatic animal, improve cultured output, and there is important science, reality and economic implications to solving the problems such as bacterial drug resistance, fishery products drug residue and water environment pollution that cause due to long-term abuse of antibiotics that face in sea farming.

Epinephelus coioide (Epinephelus coioides, orange-spotted grouper) is commonly called as blue spot, is lower floor fish in water warm, is mainly distributed in the Indian Ocean, Red sea, Mediterranean Sea, North Western Pacific and south east asia.Its delicious meat, nutritious, strong stress resistance, growth is fast, body colour is gorgeous, market value is high and stable, at present Epinephelus coioide has become the amount of growing seedlings in the world and has propagated one of important marine fish of ablen ，Ye Shi China Fujian, Guangdong, Hainan, Taiwan Si Sheng that output is the highest artificially.But in recent years, the fish disease causing due to invasive organism breaks out, make Epinephelus coioide aquaculture bearing huge financial loss (1. woods is built brightness. the biological characteristics of Epinephelus coioide and cultural technique [J]. Shandong fishery, 2008,25 (2): 22-23; 2. yellow auspicious virtue. the research [J] of Epinephelus coioide vibrio alginolyticus disease. aquatic science, 2005,24 (6): 1-3; 3. plum ice, Zhou Yongcan, Xu Xiandong, Wang Shifeng, Xie Zhenyu. the separation of Epinephelus coioide tail pathogenic bacteria and evaluation [J]. Tropical Ocean journal, 2010,29 (6): 118-124).

The applicant in early-stage Study from the green hata (Epinephelus awoara) clone obtained the hepcidin gene that 1 mature peptide has 4 cysteine residues, then from the culturing Epinephelus coioides of Zhangpu, Fujian, clone has obtained the hepcidin gene that another 1 new mature peptide contains 4 cysteine residues, there is homology with other 2 hepcidin gene EC-hepcidin1 and the EC-hepcidin2 that find that reported in Epinephelus coioide, supposition is its variant, therefore called after EC-hepcidin3.Found that the EC-hepcidin1 of synthetic and the mature peptide of EC-hepcidin2 have antibacterial, antiviral effect (4.Jing-Geng Zhou, Jing-Guang Wei, Dan Xu, Hua-Chun Cui, Yang Yan, Zheng-Liang Ou-Yang, Xiao-Hong Huang, You-Hua Huang, Qi-Wei Qin.Molecular cloning and characterization of two novel hepcidins from orange-spotted grouper, Epinephelus coioides[J] .Fish & Shellfish Immunology, 2011, 30:559-568).

The fish hepcidin structure of having found with other is the same, EC-hepcidin3 comprises the signal peptide that is positioned at N end of endoplasmic reticulum target, leading peptide and the mature peptide that is positioned at C end, wherein leading peptide and mature peptide form precursor peptide (propeptide of EC-hepcidin3, EC-proHep3) jointly.

Summary of the invention

Object of the present invention aims to provide prokaryotic expression product of a kind of Epinephelus coioides antimicrobial petide and preparation method thereof.

A kind of expression vector that contains EC-hepcidin3 gene that the present invention builds can be in intestinal bacteria abduction delivering, and obtain in vitro a large amount of expression products by efficiently purifying expression product.The described expression vector that contains EC-hepcidin3 gene is at prokaryotic expression carrier pET-28a ⁺the recombinant expression vector pET28a/EC-hepcidin3 that middle insertion EC-hepcidin3 precursor peptide gene order forms.

The expression product obtaining in vitro of the present invention is the recombinant protein EC-proHep3 that recombinant expression vector pET28a/EC-hepcidin3 obtains in intestinal bacteria after abduction delivering and purifying.

The gene order of described EC-hepcidin3 precursor peptide is as shown in sequence table the 1st sequence.

The aminoacid sequence of described EC-hepcidin3 precursor peptide is as shown in sequence table the 2nd sequence.

The preparation method of described recombinant protein EC-proHep3 comprises the following steps:

1) build recombinant expression vector;

2) by step 1) gained recombinant expression vector importing host cell, acquisition can be expressed the genetically engineered bacteria strain of EC-proHep3;

3) the genetically engineered bacteria strain that fermentation culture has obtained also carries out abduction delivering, obtains expression product;

4) purification procedures 3) expression product of gained, obtain recombinant protein EC-proHep3.

In step 1) in, described expression vector can be pET-28a ⁺deng.

In step 2) in, described host cell can be intestinal bacteria E.coli Rosette etc.

In step 3) in, described expression product is for first expressing thalline by centrifugal collection, will express the supernatant liquor that after the resuspended and ultrasonication of thalline, centrifugal collection obtains again.

In step 4) in, described separation and purification can adopt affinity chromatography purifying.

The present invention, by Protocols in Molecular Biology, utilizes prokaryotic expression carrier pET-28a ⁺with prokaryotic expression bacterial strain E.coli Rosette, recombinant protein EC-proHep3 soluble, easy purifies and separates, that have anti-microbial activity has been expressed in success in vitro.

Recombinant protein EC-proHep3 has the anti-microbial activity of wide spectrum, gram-positive microorganism, as micrococcus lysodeikticus (Micrococcus lysodeikticus Fleming), Corynebacterium glutamicum (Corynebacterium glutamicum) and streptococcus aureus (Staphylococcus aureus), is all had to obvious restraining effect to Gram-negative bacteria as Pseudomonas stutzeri (Pseudomonas stutzeri) and Pseudomonas fluorescens (Pseudomonas fluorescens).

The gene engineering expression product of recombinant protein EC-proHep3 has important using value by research and develop the aspects such as resisting pathogenic microbes new drug, fodder additives and immunostimulant in Epinephelus coioide aquaculture.

Accompanying drawing explanation

Fig. 1 is pcr amplification Epinephelus coioides antimicrobial petide Hepcidin3 precursor peptide gene order result electrophorogram.In Fig. 1,1 is pcr amplification after product; M is DL2000DNA Marker.

Fig. 2 is pET-28a ⁺carrier enzyme is cut the electrophorogram after processing.In Fig. 2, bp represents base pairs; M is DL2000DNA Marker; 1 for not carrying out the pET-28a that enzyme is cut ⁺carrier, 2 for using NcoI and XhoI to carry out the pET-28a after double digestion ⁺carrier.

Fig. 3 is for detecting the SDS-PAGE electrophorogram of protein expression situation.In Fig. 3, KDa represents the molecular weight (kilodalton) of protein; M1 is protein Marker; 1 is bacterial protein sample before the induction of pET28a/EC-hepcidin3 recombinant expression vector; 2 is bacterial protein sample after the induction of pET28a/EC-hepcidin3 recombinant expression vector.

Fig. 4 is the SDS-PAGE electrophorogram of the soluble analysis of expression product, and in Fig. 4, KDa represents the molecular weight (kilodalton) of protein; M2 is protein Marker; 1 supernatant samples for ultrasonication expression thalline centrifugal rear collection; 2 for expressing the deposit sample of thalline centrifugal rear collection for ultrasonication.

Fig. 5 is the purifying figure of affinity chromatography purification of recombinant proteins EC-proHep3.In Fig. 5, X-coordinate is the liquor capacity Volume (ml) of purification column of flowing through; Ordinate zou is for flowing out solution after the purification column ultraviolet absorption value Absorbance (mAU) under 280nm wavelength; A is for flowing out component, and b is elution fraction.

Fig. 6 is the SDS-PAGE electrophorogram after recombinant protein EC-proHep3 purifying.In Fig. 6,1 flows out component sample in purge process; 2 is elution fraction sample in purge process; M2 is protein Marker; KDa represents the molecular weight (kilodalton) of protein.

Embodiment

Be described with reference to the accompanying drawings by the following examples technical scheme of the present invention.

The structure of embodiment 1 Epinephelus coioides antimicrobial petide Hepcidin3 recombinant expression vector

1) acquisition of Epinephelus coioides antimicrobial petide Hepcidin3 precursor peptide gene

According to pET-28a ⁺multiple clone site on carrier and Epinephelus coioides antimicrobial petide Hepcidin3 gene order, the synthetic specificity of design upstream and downstream primer, pcr amplification Epinephelus coioides antimicrobial petide Hepcidin3 precursor peptide gene order.

At the 5` of upstream primer ECH-exp-S1 end, add NcoI restriction enzyme site, at the 5` of downstream primer ECH-exp-A1 end, add XhoI restriction enzyme site, terminator codon and 6 * His are histidine-tagged: downstream primer adopts XhoI restriction enzyme site:

ECH-exp-S1:5`GCG

gCTTACCCGTCACTGGAGTAG 3`, italic also marks the NcoI restriction enzyme site that underscore partly represents introducing.

ECH-exp-A1:5`CCG

tTGGAAGTCACAGCGGACTCC 3`, italic also marks the XhoI restriction enzyme site that underscore partly represents introducing, and black matrix partly represents terminator codon, is 6 * His-Tag sequence in square frame.

The Epinephelus coioides antimicrobial petide Hepcidin3cDNA of take restructuring pMD18-T positive plasmid is amplification template, take ECH-exp-S1 and ECH-exp-A1 as upstream and downstream primer, with Ex Taq enzyme (purchased from TaKaRa company), carry out pcr amplification goal gene fragment, reaction system is as shown in table 1.

Table 1

Aseptic MiliQ water	69μL
		10 * buffer is (containing Mg ²⁺)	10μL
dNTPs(2.5mM each)	8μL
		pMD 18-T/EC-Hepcidin3	4μL
Upstream primer ECH-exp-S1 (10 μ M)	4μL
		Downstream primer ECH-exp-A1 (10 μ M)	4μL
Ex Taq enzyme (5U/ μ L)	1μL
		Total reaction volume	100μL

Mix, on thermal cycler, according to following program, carry out PCR reaction: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30sec, 60 ℃ of annealing 30sec, 72 ℃ are extended 1min; Carry out after 30 circulations, 72 ℃ are extended 5min.

After reaction finishes, all reaction solutions are carried out to 1.5% (W/V) sepharose-TAE electrophoresis, reclaim test kit reclaim specific fragment with Qiagen gel, obtaining PCR product length is 236bp (referring to Fig. 1).

Get the goal gene fragment of the above-mentioned recovery of 2 μ g, with NcoI and XhoI Restriction Enzyme double digestion, reaction solution is placed in 37 ℃ of water-baths and reacts 8h.It is as shown in table 2 that enzyme is cut system.

Table 2

10×K buffer	6μL
		10×BSA	6μL
NcoI	1.5μL
		XhoI	1.5μL
Reclaim goal gene fragment	25μL
		MilliQ water	20μL
Total reaction volume	60μL

After reaction finishes, with 1.5% (W/V) sepharose-TAE electrophoresis detection enzyme, cut efficiency, coprecipitated dose of goal gene fragment reclaiming after enzyme is cut of TaKaRa nucleic acid.Goal gene fragment after processing has the sticky end of NcoI and XhoI.

2) pET-28a ⁺the processing of plasmid

By-80 ℃ of preservations with pET-28a ⁺e.colistraindh5α line on the LB flat board containing the kantlex of 50 μ g/mL, 37 ℃ of overnight incubation.Next day, picking mono-clonal bacterium colony, was inoculated in the LB liquid nutrient medium of 5mL containing the kantlex of 50 μ g/mL, and 37 ℃, 200rpm/min cultivates 8h to logarithmic phase, and centrifugal collection thalline extracts pET-28a in a small amount ⁺plasmid.

Get the pET-28a after said extracted ⁺plasmid, with NcoI and XhoI Restriction Enzyme double digestion, reaction solution is placed in 37 ℃ of water-baths and reacts 8h.It is as shown in table 3 that enzyme is cut system.

Table 3

10×K buffer	6μL
		10×BSA	6μL
NcoI	1.5μL
		XhoI	1.5μL
pET-28a ⁺Plasmid	25μL
		MilliQ water	20μL
Total reaction volume	60μL

After reaction finishes, with 1.0% (W/V) sepharose-TAE electrophoresis detection enzyme, cut efficiency (referring to Fig. 2), coprecipitated dose of plasmid reclaiming after enzyme is cut of TaKaRa nucleic acid.Plasmid after processing has the sticky end of NcoI and XhoI.

3) structure of pET28a/EC-hepcidin3 recombinant expression vector, conversion and evaluation

The pET-28a of the sticky end of NcoI and XhoI will be there is after a series of processing ⁺plasmid with there is identical sticky end goal gene fragment and connect, reaction system is as shown in table 4.

Table 4

pET-28a ⁺Plasmid	1μL
		Goal gene fragment	4μL
Ligation Solution I	5μL
		Total reaction volume	10μL

Reaction solution is placed in to 16 ℃ of connections and spends the night, next day, get 5 μ L ligation liquid and be converted in 50 μ l E.coli DH5 α competent cells, be coated on overnight incubation on the LB solid medium flat board that contains kantlex (50 μ g/ml).The positive bacterium colony of picking next day, take ECH-exp-S1 and ECH-exp-A1 as upper, downstream primer, by PCR method, identify positive colony bacterium as previously mentioned, by Shanghai, Sheng Gong company carries out DNA nucleotide sequence mensuration, result shows and connects correctly, the open reading frame coding of Nucleotide continuously, obtain pET28a/EC-hepcidin3 recombinant expression vector, this recombinant expression vector adopts T7 promotor, the N end of recombinant protein EC-proHep3 contains 1 methionine residues and 1 glycine residue, C end contains 6 * His-Tag, be convenient to adopt the method purifying target protein of affinity chromatography.

The embodiment abduction delivering of 2 pET28a/EC-hepcidin3 recombinant expression vectors in intestinal bacteria and the soluble analysis of expression product

1) abduction delivering

PET28a/EC-hepcidin3 recombinant expression vector is converted in E.coli Rosette competent cell, and coats on the LB solid medium flat board that contains 50 μ g/ml kantlex 37 ℃ of overnight incubation.Next day, picking mono-clonal bacterium colony, was inoculated in the LB liquid nutrient medium that 20ml contains 50 μ g/ml kantlex, and 200rpm, 37 ℃ of shaking culture are to OD ₆₀₀be about 0.3～0.4, then add IPTG to final concentration be 0.4mM, at 30 ℃, 180rpm shaking culture 6h.Get each 1ml of bacterium liquid of IPTG induction front and back, centrifugal collection thalline, utilizes polyacrylamide gel electrophoresis (SDS-PAGE) to detect the expression (referring to Fig. 3) of albumen.Result shows that pET28a/EC-hepcidin3 recombinant expression vector has obvious protein band being less than 19kDa place before induction in E.coli Rosette after IPTG induction.

2) soluble analysis

By the centrifugal collection thalline of bacterium liquid after induction, PBS solution (pH8.0) suspension thalline with precooling, after-20 ℃ of freeze thawing 1 time, ultrasonic disruption somatic cells under condition of ice bath, ultrasonication liquid is centrifugal and collect respectively supernatant liquor and deposit sample at 4 ℃, utilize SDS-PAGE electrophoresis, carry out the soluble analysis (referring to Fig. 4) of expression product.Result shows that target protein matter is solubility expression, and size is about 10kDa.

Embodiment 3 affinity chromatography purification of recombinant proteins EC-proHep3

1) prepare metal chelating chromatography column

Metal chelate affinity chromatography filler is sepharose fast flow (GE Healthcare).

Chromatography reagent is:

Solution A: 200mmol/L single nickel salt;

Solution B: 25mmol/L NaH ₂pO ₄, 500mmol/L NaCl, pH 4.0 (adjusting with phosphoric acid);

Solution C: 50mmol/L phosphoric acid buffer, 200mmol/L NaCl, 50mmol/L imidazoles, pH 8.0;

Solution D: 50mmol/L phosphoric acid buffer, 150mmol/L NaCl, 400mmol/L imidazoles, pH 8.0.

All solution is all used after 0.45 μ m membrane filtration, stand-by.

After affinity chromatography medium dress post, according to following steps, operate:

Solution A chelating Ni ²⁺; Ultrapure washing post; Solution B washes away unnecessary Ni ²⁺; Ultrapure washing post; The abundant balance chromatography column of solution C.The liquor capacity using in every step is all about 5 column volumes.So far, metal chelate affinity chromatography post is ready to, stand-by.

2) preparation of upper prop sample

According to the description in embodiment 2, collect the supernatant liquor after ultrasonication, and through 0.45 μ m membrane filtration, treat column purification.

3) upper column purification

By the whole upper props of ready sample, and collect after outflow component, first by the solution C of 5～10 column volumes, wash away unconjugated foreign protein; By imidazoles gradient, further wash away the foreign protein that may have some non-specific binding again; Finally use solution D wash-out target protein, and collect elution fraction (referring to Fig. 5).

Take a morsel and flow out component and elution fraction carries out SDS-PAGE electrophoresis evaluation (referring to Fig. 6).Result demonstration, EC-proHep3 recombinant protein can be specificly combined with nickel ion chelating affinity column, and after imidazoles solution (400mM) the competition washing of higher concentration, fusion expressed product is by wash-out, and purity is higher.

The elutriant that contains target protein is packed in the dialysis band of anticipating, under cold condition, put into Tris-Cl damping fluid (50mM Tris, 100mM NaCl, pH 8.0) and dialyse, repeatedly change dialyzate.Finally dialyse (20mM Tris, 20mM NaCl, pH 8.0) in Tris-Cl damping fluid.

The mensuration of embodiment 4 recombination expression product EC-proHep3 anti-microbial activities

1) application Bradford method is measured the concentration of recombination expression product EC-proHep3

Get 10 times of 1 * PBS dilutions for BSA liquid storage, final concentration is 0.5mg/ml; 0.5mg/ml BSA standard substance are added in 96 porocyte culture plates by 0,1,2,4,8,12,16,20 μ l gradients, with 1 * PBS, complement to respectively 20 μ l, each concentration gradient arranges 3 Duplicate Samples; Every hole adds 200 μ l G250 staining fluids, and room temperature is placed 3min; By microplate reader, measure the absorbancy at 595nm wavelength place; Obtain protein concentration typical curve.

Application Bradford method is measured the protein concentration typical curve that BSA standard substance obtain, and can calculate the concentration of recombinant expression protein according to formula.Use sterilizing ultrapure water by protein solution doubling dilution to 1.5～96 μ M.

2) mensuration of recombination expression product EC-proHep3 anti-microbial activity

MIC (Minimum Inhibitory Concentration, minimal inhibitory concentration) mensuration is with reference to method (the 5.Bulet P of Bulet etc., Dimarcq J L, Hetru C, Lagueux M, Charlet M, Hegy G, Van Dorsselaer A, Hoffmann J A.A novel inducible antibacterial peptide of Drosophila carries an O-glycosylated substitution[J] .J.Biol.Chem., 1993,268:14893-14897; 6.Ke-Jian Wang, Jing-Jing Cai, Ling Cai, Hai-Dong Qu, Ming Yang, Min Zhang.Cloning and expression of a hepcidin gene from a marine fish (Pseudosciaena crocea) and the antimicrobial activity of its synthetic peptide[J] .Peptides., 2009,30 (4): 638-646), on 96 porocyte culture plates, carry out.

(1) preparation of bacteria suspension: get tested bacterium, line nutrient broth flat board, under optimal temperature, cultivate 12～16h; 2～3 clones of picking are inoculated in MH inclined-plane, continue to cultivate 12～16h, reach the logarithmic phase of bacterium; 10mM sodium phosphate salt damping fluid (pH 72) rinses the MH bacterium slant culture of fresh preparation, and bacteria suspension adjustment is diluted to OD ₆₀₀=0.1, get 18 μ lOD ₆₀₀=0.1 bacteria suspension add final volume be 1ml MH diluted medium (PBS: MH=3: 2, V/V) in, this bacteria suspension is MIC working concentration.

(2) every kind of tested bacterium is according to following operation setting blank group, negative control group and testing sample experimental group, and every group arranges 2 Duplicate Samples, and every kind of bacterium is repeated 3 times:

1. positive controls: add 50 μ L sterilizing ultrapure waters and 50 μ L bacteria suspensions;

2. blank group: add 50 μ L testing protein samples and 50 μ L sodium phosphate salt damping fluids;

3. sample experimental group: add 50 μ L testing protein samples and 50 μ L bacteria suspensions;

(3), after the suitableeest cultivation 24～48h of bacterium, observe MIC result, as shown in table 5.

Table 5

Strain name	CGMCC No	MIC(μM)
			Gram-positive
Corynebacterium glutamicum	1.1886	48～96
			Micrococcus lysodeikticus	1.0634	24～48
Streptococcus aureus	1.0363	1.5～3
			Gram-negative
Pseudomonas fluorescens	1.0032	24～48
			Pseudomonas stutzeri	1.1803	＜1.5

CGMCC No.: China Committee for Culture Collection of Microorganisms's common micro-organisms center bacterium numbering;

MIC: minimal inhibitory concentration, with (a)～(b) expression, (a) represent the maximum concentration of the visible thalli growth of naked eyes, (b) represent to lose the Cmin of thalli growth.

Claims

1.EC-hepcidin3 the gene order of precursor peptide, is characterized in that, as shown in sequence table the 1st sequence.

2.EC-hepcidin3 the aminoacid sequence of precursor peptide, is characterized in that, as shown in sequence table the 2nd sequence.

3. the preparation method of recombinant protein EC-proHep3, is characterized in that comprising the following steps:

1) build the recombinant expression vector of the gene order that contains EC-hepcidin3 precursor peptide as claimed in claim 1;

2) step 1) gained recombinant expression vector is imported to host cell, acquisition can be expressed the genetically engineered bacteria strain of EC-proHep3;

4. the preparation method of recombinant protein EC-proHep3 as claimed in claim 3, is characterized in that in step 1), and described expression vector is pET-28a ⁺.

5. the preparation method of recombinant protein EC-proHep3 as claimed in claim 3, is characterized in that in step 2) in, described host cell is intestinal bacteria E.coli Rosette.

6. the preparation method of recombinant protein EC-proHep3 as claimed in claim 3, is characterized in that in step 3), and described expression product is for first expressing thalline by centrifugal collection, will express the supernatant liquor that after the resuspended and ultrasonication of thalline, centrifugal collection obtains again.

7. the preparation method of recombinant protein EC-proHep3 as claimed in claim 3, is characterized in that in step 4), and described separation and purification adopts affinity chromatography purifying.