CN101386642B - Expression of vibrio alginolyticus outer membrane protein VA0760 and application thereof as vaccine component - Google Patents
Expression of vibrio alginolyticus outer membrane protein VA0760 and application thereof as vaccine component Download PDFInfo
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- CN101386642B CN101386642B CN2008100298335A CN200810029833A CN101386642B CN 101386642 B CN101386642 B CN 101386642B CN 2008100298335 A CN2008100298335 A CN 2008100298335A CN 200810029833 A CN200810029833 A CN 200810029833A CN 101386642 B CN101386642 B CN 101386642B
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Abstract
The invention relates to the expression of a recombined outer membrane protein VA0760 of Vibrio alginolyticus and the action function of a vaccine component. Through cloning, expressing and purifying genes of the recombined outer membrane protein VA0760 of the Vibrio alginolyticus, a purified product can obviously improve the resistance of the fish to pathogenic bacteria such as Vibrio alginolyticus, pseudomonas fluorescens. As a target candidate of a subunit vaccine, the recombined outer membrane protein VA0760 of the Vibrio alginolyticus has great significance on the immunological prevention and treatment of controlling Vibrio infection.
Description
Technical field
The present invention relates to the immunoloregulation function of a kind of outer membrane protein (outer membrane protein, OM protein) VA0760 of vibrio alginolyticus (Vibrio alginolyticus) expression.
Background technology
Vibrio alginolyticus is distributed widely in seawater and place, river mouth all over the world, and quantity occupies first of the seawater class vibrios, for the vibrio alginolyticus pathogenic microorganism; The emphasis of research is exactly an immune analysis, illustrates its pathogenesis and finds out the effectively preventing method, and the way of control comprises that mainly medicine is microbiotic control and immune protection; Though wherein microbiotic has been obtained huge progress in initial use; Yet be widely used along with antibiotic, a series of associated problem also occurred, antibiotic unreasonable use on the one hand; Increase the generation of antibiotic resistant bacteria, infectation of bacteria increases; On the other hand, because of antibiotic resistance escalated dose artificially, aggravate chemical sproof generation.Therefore, development is that component vaccines has broad application prospects to have in immanoprotection action efficient with antigen.
In the control of various Animal diseases, the use of vaccine is one and has low cost, high-efficiency method, and had the part vaccine to be used on a large scale and obtained good control effect.The vaccine kind is a lot, is example with the bacterium, comprises whole-bacterial-vaccine, subunit vaccine and dna vaccination etc.From the situation of practical application, though whole-bacterial-vaccine has the simple relatively and also lower advantage of cost of making, also there are much some shortcomings of inherent owing to use whole cell in it.Like the attenuation whole-bacterial-vaccine adverse consequencess such as toxicity recovery that cause bacterial strain sudden change possibly appear and; The deactivation whole-bacterial-vaccine is then often owing to the inactivation process to bacterium causes the active change of some immunoprotection groups, and influences the effect of immunoprotection.And concerning whole-bacterial-vaccine, because the complicacy of bacterium composition, wherein often have some toxic components (like LPS etc.), can cause body to produce untoward reaction.And the conventional whole-bacterial-vaccine immune protective effect that makes is undesirable.Yet whole-bacterial-vaccine remains main vaccine kind at present.Its reason possibly and identify that difficulty is relevant with the antigenic discovery of efficient neutralization.
Along with genomics, proteomics with transcribe the development of distribution plan technology, this just provides new opportunity for bacterial vaccine.Recombinant vaccine becomes the emphasis of vaccine research owing to the various shortcoming that has efficiently and broken away from traditional vaccine, has wide applicating and exploitation prospect.Outer membrane protein is one of staple of Gram-negative bacteria adventitia; Because outer membrane protein is directly faced host's body fluid and tissue; Physiological activities such as bacterium utilizes that outer membrane protein sticks, intrusion and inflammation, more easily by host's identification as target of attack, make the outer membrane protein of Gram-negative bacteria have good immunogenicity; Not only can stimulate humoral immunization, and the pair cell immunity also there is hormesis.In recent years, some outer membrane proteins of bacterium have been proved has good immunogenicity, has good prospect with its preparation recombinant vaccine.As in intestinal bacteria, finding the immunity protection function of outer membrane protein OmpX.But before the present invention comes forth, any immunoloregulation function that discloses or reported the vibrio alginolyticus outer membrane protein VA 0760 of mentioning in the present patent application is not arranged as yet.
This patent takes the lead in adopting modern biologies such as molecular cloning and immunology technology, is research object with the vibrio alginolyticus, and external membrane protein gene VA0760 clones, expression and purifying, and furthers investigate its immune protective on this basis.Immunity with attack the outer membrane protein V A0760 that malicious experimental result shows vibrio alginolyticus and demonstrate the immanoprotection action higher vibrio alginolyticus.Its premunition protection ratio (RPS) is 75%, demonstrates the cross immunity originality to Pseudomonas fluorescens in addition, and its immune protective rate (RPS) reaches 52.6%.These presentation of results VA0760 can be used as vaccine component.
Summary of the invention
Outer membrane protein has good immunogenicity, and pair cell immunity and humoral immunization all have hormesis.The present invention relates to find to significantly improve resistance power behind the immune crucian to important pathogenic bacteria vibrio alginolyticus and Pseudomonas fluorescens through to the vibrio alginolyticus outer membrane protein VA 0760 immune protective of expression and the analysis of immune intercrossing.Its feasibility of preliminary assessment as candidate vaccine, significant to the control of aquatic products disease.
Description of drawings
The 0.8% agarose gel electrophoresis analysis of Fig. 1 .VA0760 gene PCR product
The double digestion of Fig. 2 .pET-28a-VA0760 recon is identified
Fig. 3. clone the abduction delivering of sub-BL21-pET-28a-VA0760 in E.coli-BL21
Fig. 4 .SDS-PAGE analyzes the recombinant protein lane 1 of purifying: clone sub-BL21-pET-28a-VA0760 abduction delivering supernatant; Lane 2: purified recombinant albumen
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to normal condition; The condition described in the Sambrook equimolecular cloning experimentation chamber handbook (2001 by Cold Spring Harbor Laboratory Press) for example, or the condition of advising according to manufacturer.
The clone of vibrio alginolyticus outer membrane protein VA 0760 gene and evaluation
1. the amplification of vibrio alginolyticus VA0760 gene
Because the complete sequence of vibrio alginolyticus gene is undetermined also, there is not the VA0760 gene order report of this bacterium at present yet.Accomplished complete sequence determination in view of Vibrio parahaemolyticus, and announced at DB GenBank (BA000031) in March, 2006, we by the VP0760 sequences Design of having announced on its DB the VA0760 gene primer, be used for the pcr amplification of vibrio alginolyticus VA0760.Institute's designed primer is: upstream primer: 5 '-ATA
GGATCCATGTCTTACCTAAAGAAAAGCC-3 '; Downstream primer: 5 '-CGC
AAGCTTTTAGAAGTAGTATTCAAC-3 '; Horizontal line is depicted as restriction enzyme site, and upstream primer is introduced the BamHI restriction enzyme site, and downstream primer is introduced the HindIII restriction enzyme site.Use bacterial genomes DNA extraction test kit (TIANGEN company), the complete genome DNA that extracts vibrio alginolyticus according to the working instructions in the test kit adopts above-mentioned primer to carry out pcr amplification, pcr amplification result shown in Figure 1 as template.Visible from scheming, gene amplification product is single specific band, and the about 1119bp of VA0760 gene is suitable with the clip size of estimating.
2, the clone of vibrio alginolyticus VA0760 gene, screening and evaluation
Use 0.8% agarose/ethidium bromide gel to carry out electrophoresis the gene PCR product, through the agarose gel electrophoresis purifying of new configuration, the specification sheets that reclaims test kit (DNA Gel Extraction Kit) by the gel available from TIANGEN company reclaims.With clean, sharp blade, under short wavelength ultravioletlamp, downcut the purpose pcr amplified fragment, excise unnecessary glue as much as possible, the centrifuge tube of place totally, having weighed is weighed and is write down the weight of downcutting purpose glue; To specifications step then, add successively various solution, centrifugal, cross post, wash-out etc., obtain gene PCR at last and reclaim product, place-20 ℃ of preservations.Take a morsel and carry out the recovering effect electrophoresis detection.
After treating that the gene PCR amplified production reclaims; Adopt corresponding restriction enzyme BamHI and HindIII (Takara company) to carry out double digestion respectively; Simultaneously carrier pET-28a (Novagen company) is carried out double digestion (37 ℃ of enzymes are cut and spent the night), enzyme is cut the back and is used ethanol sedimentation to reclaim.Method is following: add the NaAc (3M, pH 5.2) of 1/10 volume and the absolute ethyl alcohol of 2.5 times of volumes to the enzyme system of cutting, through-20 ℃ leave standstill 60min after; The centrifugal 15min of 12000rpm; Abandon supernatant, add 500
170% washing with alcohol, the centrifugal 5min of 12000rpm; Abandon supernatant; Repeated washing is once use up liquid carefully after having washed, exhaust ethanol with filter paper; Put natural air drying in the super clean bench, directly be dissolved in an amount of de-ionized sterilized water after air-dry.
Both are reclaimed the back add ligase enzyme (Takara company), regulate 16 ℃ of water temperatures, connect afterwards and spend the night.To connect product mixes with about 100 μ lDH5 α competent cells; 42 ℃ of heat shock 90s behind the ice bath 30min; And ice bath 5min immediately, add fresh LB substratum and supply volume to 1ml, place 37 ℃ of shaking table 150rpm slowly to cultivate 45min; Get the centrifugal 30s of nutrient solution 10000rpm; Abandon 850 μ l supernatants, remaining bacterium liquid is resuspended evenly, and coating contains kantlex (100
g/ml) the LB flat board; After liquid to be transformed is absorbed by flat board fully, be inverted cultivation 12～16 hours in 37 ℃.
6 single bacterium colonies of picking at random from transform flat board are inoculated into 5ml and add in the LB liquid nutrient medium of kantlex (50mg/ml) in 1: 500 ratio, cultivate 8h for 37 ℃, adopt alkaline lysis to extract plasmid in a small amount.Method is following: bacterium liquid is changed in the 1.5mL Eppendorf tube; 4 ℃ of centrifugal 1min of 10000g, the sucking-off nutrient solution adds solution I (the 50mmol/L glucose that 100 μ L ice precooling; 25mmol/L Tris-Cl pH 8.0,10mmol/L EDTA) thermal agitation to bacterial precipitation is resuspended fully.Static 2min under the room temperature, add the solution II that 200 μ L join at present (0.2mmol/LNaOH, 1%SDS); Cover the tight mouth of pipe, slightly put upside down mixing several times, ice bath 5min fast up and down; The solution III (60mL 5mol/L potassium acetate, 11.5mL glacial acetic acid, the 28.5mLH that add the precooling of 150 μ L ice
2O), put upside down repeatedly the heavy-gravity bacterial lysate is uniformly dispersed, ice bath 5min.4 ℃ of centrifugal 7min of following 12000g shift 350 μ L supernatants to another centrifuge tube, with the phenol of equivalent volumes/chloroform extracting 2 times, add the ice-cold absolute ethyl alcohol of 2 times of volumes, and mixing turns upside down.Room temperature leaves standstill 2min, 4 ℃ of centrifugal 5min of 12000g.Carefully remove supernatant, 75% washing with alcohol 2 times is abandoned supernatant, and the drop that will invest on the tube wall again eliminates, and the TE (pH8.0) that contains the RNA enzyme with 30 μ L dissolves DNA, and vibration is stored in-20 ℃ a little.Carrying out double digestion with corresponding restriction enzyme further identifies.As shown in Figure 2, but recombinant plasmid pET-28a-VA0760 enzyme cut out and the consistent fragment of PCR product sizableness clip size, show that gene successfully is inserted in the pET-28a carrier.The recombinant plasmid VA0760 that utilization Beckman CEQ end mark cycle sequencing will have been cloned carries out sequencing.
The proteic expression of vibrio alginolyticus VA0760
1 predicted amino acid sequence
Gene VA0760 is the moderate-length fragment, and utilization DNAssist software analysis, obtains its complete ORFs ORF
The prokaryotic expression of 2 recons
Single colony inoculation of picking recombinant plasmid adds in the LB liquid nutrient medium of kantlex (50mg/ml) 37 ℃ of incubated overnight to saturated in 1ml in 1: 500 ratio; In the LB substratum of 5mL, cultivate about 2h to OD by 1: 100 inoculum size inoculation saturated culture in 37 ℃
600Approximate about 0.6.In culture, adding IPTG is 1mmol/L to final concentration, and 37 ℃ are continued to cultivate 3h.Take out through inductive culture room temperature high speed centrifugation 1min, collect thalline.Set up control group simultaneously, deposition is resuspended among 100 μ L, 2 * sds gel application of sample buffer, 100 ℃ of heating 5min.12000rpm, centrifugal 10min.Get an amount of going up and please be splined on the 12%SDS polyacrylamide gel by sample, electrophoresis to bromjophenol blue is moved to the separation gel bottom.Coomassie brilliant blue staining, scanning output photo.The result shows that VA0760 recombination has changed E.coli BL21 over to and obtained positive expression such as Fig. 3.Its recombinant protein size is about 41kD.Therefore the molecular weight of recombinant protein conforms to expection on expression map.
Embodiment 3
The proteic purifying of vibrio alginolyticus VA0760
Picking recombinant plasmid transformed e. coli bl21 (DE3), according to corresponding carrier pET-28a, respectively in the LB substratum that contains 100 μ g/mL kantlex, 37 ℃ of overnight cultures.Picking list bacterium colony adds 5mL LB liquid in 37 ℃ of shaken overnight immediately, is forwarded in 1: 100 ratio to contain antibiotic liquid LB substratum 200mL, 37 ℃ of about 2.5-3h of shaking culture, OD
600=0.6 o'clock, in bottle, add IPTG (final concentration is 1mmol/L) and induce, 30 ℃ of 200r/min shake 3h.6000g, 4 ℃ of centrifugal 10min collect thalline, wash thalline 2 times with saline water, claim the thalline weight in wet base.
The thalline of collecting is dissolved in buffer D (8M urea with 1: 100 (g/mL); 0.1MNaH
2PO
410mmTris-HCl, pH 8.0) the suspension thalline, ultrasonication, output rating 60%, 5s/ time, interval 9s, ultrasonic 30min.Bacterium liquid after the ultrasonication, 9000g, 4 ℃ of centrifugal 15min collect supernatant.Ultrasonic centrifugal supernatant carries out purifying with the Ni-NTA affinity column.Adopt the damping fluid purifying protein of pH5.9 (buffer F) and two different pH values of pH4.5 (buffer G).With dcq buffer liquid bufferE (8M urea; 0.1M NaH
2PO
410mmTris-HCl, pH 6.3) wash each 1mL 50 times; With elution buffer buffer F (8M urea; 0.1M NaH
2PO
410mmTris-HCl, pH 5.9) wash-out target protein 4 times, each 1mL collects elutriant; With elution buffer buffer G (8M urea; 0.1M NaH
2PO
410mmTris-HCl, pH 4.5) eluted protein 4 times, each 1mL collects elutriant.Get liquid collecting and carry out the SDS-PAGE electrophoresis result and see Fig. 4, Bradford protein quantification standard measure, cryopreservation is subsequent use.
Embodiment 4
Vibrio alginolyticus outer membrane protein VA 0760 is total to 1.0mg to the immanoprotection action purifying outer membrane protein V A0760 of vibrio alginolyticus and Pseudomonas fluorescens; Crucian is carried out abdominal injection; The antigen amount of each immunity be 25
the g/ tail, every group of totally 20 tails.First immunisation is used Freund's complete adjuvant emulsification, and at a distance from two all booster immunizations once, booster immunization is used Freund's incomplete adjuvant emulsification.The while control group is injecting normal saline then.And the crucian to health carries out vibrio alginolyticus and Pseudomonas fluorescens mld LD
50Mensuration, according to waiting logarithmic interval to select experimental concentration.Behind booster immunization the 10th day, use 5 times of LD
50The vibrio alginolyticus (5 * 10 of dosage
7Cfu/mL) and Pseudomonas fluorescens (5 * 10
7Cfu/mL) attack malicious protectiveness experiment.Abdominal injection is attacked the 2nd day behind the poison, and moving about of control group fish begins to become slow, and constantly dead, and has the no abnormal reaction of VA0760 experimental group of immanoprotection action.Fish is dead no longer successively after 5 days; Observed and recorded experimental group and control group are to death condition; The control group survival rate of injection vibrio alginolyticus and Pseudomonas fluorescens is respectively 20% and 5%; And the immune protective effect of experimental group VA0760 has tangible difference, and the immune protective rate (RPS) of vibrio alginolyticus up to 75%, is had significant difference (P＜0.01).VA0760 also demonstrates the cross-protection to Pseudomonas fluorescens simultaneously, and its immune protective rate (RPS) is 52.6% (P＜0.05).Show that through statistical calculations VA0760 has significant immanoprotection action.Explaining that VA0760 can stimulate body to produce protection antibody, improve the host immune protective capability, is that a good immunoprotection is former.
Sequence table
< 110>Zhongshan University
< 120>expression of vibrio alginolyticus outer membrane protein VA 0760 and as the application of vaccine component
<160>2
<210>1
<211>1122
<212>DNA
< 213>vibrio alginolyticus
<400>1
atgtcttacc taaagaaaag cctacttgcc acggcaatta ccggcatgat gttcagtggt 60
gttgcatttg ctgatggtgc aaacagtgac gcagcaaaag agtttctaac caaagattca 120
ttttcttatg aagtttacgg gatcatcgcg atgcaggccg cgtaccgcga ttacgattct 180
ggcagcaaag caacggacga tgacttgggt ggcatgcagc taaacaacga atctcgtatc 240
ggtttccgtg gtaagaaaca atttgctaac ttcgacccta cttttatttg gcaaatcgaa 300
ggcggttatg tagaccctag ctttggtggc gagggcgcag gtcttggtga acgtgacaca 360
ttcgtcggtt ttgaaagtgc atcttggggg caaattcgcc tgggtcgcgt tttgacacct 420
atgtacgaat tggttgactg gcctgcatct aaccctggcc tgggcgatgt atacgactgg 480
ggtggtgcta ttggtggcgc caagtaccaa gaccgtcaat caaacactat ccgctgggac 540
tctccaatgt ttgctgacaa attctcccta gatatcgcag ctggtgcagg tgataaagca 600
ggtctaggtg aaggggatga ctactggggt ggtatcgctg cacactacaa aattggtcct 660
atccagttag atgcggcata tgaaggtaac cgtaatatca agatggaaag ccaaacgtgg 720
gaaaacaaca cgtacttggt aggggcacaa ggctggtttg ataacggaat ctctttcttc 780
gcgcaataca aatacatgga ggcggacgca agcaatggtg tgagtgaaaa gcaagatgca 840
atgtctgccg ctatcatgta caccacgggt gattggcaat acaaactggc ttacgccgct 900
aactttgatc tagagcgtga tggtaagaaa attaacgata cagcggacga tgtgttatca 960
gcacaagtta tgtatttcgt agacccatcg gcagtgctgt acgtacgtgc tcgtactcta 1020
gactttggcg acggtgcgtc tcaattagat aaaccaacag aagcacgttg gaagtctgct 1080
gactacgacg agttctctgt aggtgttgaa tactacttct aa 1122
<210>2
<211>378
<212>PRT
< 213>vibrio alginolyticus
<400>2
Met Ser Tyr Leu Lys Lys Ser Leu Leu Ala Thr Ala Ile Thr Gly Met
1 5 10 15
Met Phe Ser Gly Val Ala Phe Ala Asp Gly Ala Asn Ser Asp Ala Ala
20 25 30
Lys Glu Phe Leu Thr Lys Asp Ser Phe Ser Tyr Glu Val Tyr Gly Ile
35 40 45
Ile Ala Met Gln Ala Ala Tyr Arg Asp Tyr Asp Ser Gly Ser Lys Ala
50 55 60
Thr Asp Asp Asp Leu Gly Gly Met Gln Leu Asn Asn Glu Ser Arg Ile
65 70 75 80
Gly Phe Arg Gly Lys Lys Gln Phe Ala Asn Phe Asp Pro Thr Phe Ile
85 90 95
Trp Gln Ile Glu Gly Gly Tyr Val Asp Pro Ser Phe Gly Gly Glu Gly
100 105 110
Ala Gly Leu Gly Glu Arg Asp Thr Phe Val Gly Phe Glu Ser Ala Ser
115 120 125
Trp Gly Gln Ile Arg Leu Gly Arg Val Leu Thr Pro Met Tyr Glu Leu
130 135 140
Val Asp Trp Pro Ala Ser Asn Pro Gly Leu Gly Asp Val Tyr Asp Trp
145 150 155 160
Gly Gly Ala Ile Gly Gly Ala Lys Tyr Gln Asp Asg Gln Ser Asn Thr
165 170 175
Ile Arg Trp Asp Ser Pro Met Phe Ala Asp Lys Phe Ser Leu Asp Ile
180 185 190
Ala Ala Gly Ala Gly Asp Lys Ala Gly Leu Gly Glu Gly Asp Asp Tyr
195 200 205
Trp Gly Gly Ile Ala Ala His Tyr Lys Ile Gly Pro Ile Gln Leu Asp
210 215 220
Ala Ala Tyr Glu Gly Asn Arg Asn Ile Lys Met Glu Ser Gln Thr Trp
225 230 235 240
Glu Asn Asn Thr Tyr Leu Val Gly Ala Gln Gly Trp Phe Asp Asn Gly
245 250 255
Ile Ser Phe Phe Ala Gln Tyr Lys Tyr Met Glu Ala Asp Ala Ser Asn
260 265 270
Gly Val Ser Glu Lys Gln Asp Ala Met Ser Ala Ala Ile Met Tyr Thr
275 280 285
Thr Gly Asp Trp Gln Tyr Lys Leu Ala Tyr Ala Ala Asn Phe Asp Leu
290 295 300
Glu Arg Asp Gly Lys Lys Ile Asn Asp Thr Ala Asp Asp Val Leu Ser
305 310 315 320
Ala Gln Val Met Tyr Phe Val Asp Pro Ser Ala Val Leu Tyr Val Arg
325 330 335
Ala Arg Thr Leu Asp Phe Gly Asp Gly Ala Ser Gln Leu Asp Lys Pro
340 345 350
Thr Glu Ala Arg Trp Lys Ser Ala Asp Tyr Asp Glu Phe Ser Val Gly
355 360 365
Val Glu Tyr Tyr Phe
370
Claims (3)
1. the application of the vibrio alginolyticus outer membrane protein VA 0760 of an aminoacid sequence shown in SEQ ID NO:2 in the vaccine that the anti-Pseudomonas fluorescens of preparation infects.
2. the application of the vibrio alginolyticus outer membrane protein VA 0760 of an aminoacid sequence shown in SEQ ID NO:2 in the immune protective agent that the anti-Pseudomonas fluorescens of preparation infects.
3. the application of the vibrio alginolyticus outer membrane protein VA 0760 of an aminoacid sequence shown in SEQ ID NO:2 in the raising host immune function preparation that the anti-Pseudomonas fluorescens of preparation infects.
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| CN101747416B (en) * | 2010-01-05 | 2012-11-14 | 安徽农业大学 | B-cell antigenic multi-epitope peptide linked in tandem in OmpU of vibrio mimicus, making method and application thereof |
| CN102406948A (en) * | 2011-12-05 | 2012-04-11 | 泰山医学院 | Constructing method of recombinant DNA (deoxyribonucleic acid) nucleic acid vaccine of vibrio alginolyticus HSP70 |
| CN105566461B (en) * | 2015-12-25 | 2018-12-18 | 中山大学 | Bacterial outer membrane proteins ompAs-19 after DNA reorganization and its application as immunomodulator |
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| CN1699573A (en) * | 2005-02-17 | 2005-11-23 | 厦门大学 | V. alginolyticus outer-membrane protein W gene, process for preparing the same and uses thereof |
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Non-Patent Citations (3)
| Title |
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| Polz M et al.ZP_01260826.1.《NCBI》.2006,全文. * |
| 吴灶和等.溶藻弧菌外膜蛋白对凡纳滨虾免疫功能的影响.《热带海洋学报》.2005,第24卷(第6期),全文. * |
| 黄志坚等.溶藻弧菌外膜蛋白（Va-OMP）的免疫原性及免疫保护性.《水产学报》.2006,第30卷(第4期),全文. * |
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