CN101703767B - Preparation method of nibrio parahaemolyticus outer membrane protein VP2850and application of immunity protective function thereof - Google Patents
Preparation method of nibrio parahaemolyticus outer membrane protein VP2850and application of immunity protective function thereof Download PDFInfo
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Abstract
The invention discloses a preparation method of vibrio parahaemolyticus outer membrane protein VP2850, immunity protective function and application thereof as an immune component and an antibacterial agent. Experiments show that vibrio parahaemolyticus outer membrane protein VP2850 has obvious immunity protective function, can effectively resist infections caused by vibrio parahaemolyticus, vibrio aloginolyticus, pseudomonas fluorescens and aeromonas hydrophila as well as prevent and treat diseases caused by the bacteria infections.
Description
Technical field
The present invention relates to the method for preparing of vibrio parahaemolyticus outer membrane protein VP2850 and the application of immunity protection function thereof.
Background technology
Vibrio parahaemolyticus (Vibrio parahaemolyticus) is one of vibrio common in the sea water, is to cause the pathogen that microorganism property alimentary toxicosis ranks first in recent years, also is a kind of important pathogenic bacteria in the aquaculture.Control to this type pathogenic bacterium comprises that mainly medicine is antibiotic control and immune protection.Though antibiotic has been obtained huge progress in initial use, yet be widely used along with antibiotic, a series of relevant issues also occurred, antibiotic unreasonable use on the one hand increases the generation of antibiotic resistant bacteria, and bacterial infection increases; On the other hand, because of antibiotic drug 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 a low cost, high-efficiency method, and has had the part vaccine to be used on a large scale and obtained good prevention effect.The vaccine kind is a lot, is example with the antibacterial, comprises whole-bacterial-vaccine, subunit vaccine and DNA vaccine etc.Situation from practical application; Though whole-bacterial-vaccine has the simple relatively and also lower advantage of cost of making; But also exist many owing to use whole cell more inherent shortcomings, the bacterial strain sudden change possibly occur and cause adverse consequences such as toxicity recovery like the attenuation whole-bacterial-vaccine; The deactivation whole-bacterial-vaccine then often causes some immunoprotection group activity changes owing to antibacterial being carried out deactivation, influences the effect of immunoprotection.And concerning whole-bacterial-vaccine, because the complexity of antibacterial composition, wherein often have some toxic components (like LPS etc.), can cause body to produce untoward reaction.The whole-bacterial-vaccine immune protective effect that routine makes is unsatisfactory, yet whole-bacterial-vaccine remains main vaccine kind at present, and its reason possibly and identify that difficulty is relevant with the antigenic discovery of efficient neutralization.
Genomics, proteomics and transcribe the development of scattergram technology are for bacterial vaccine provides new opportunity.Recombinant vaccine becomes the emphasis of vaccine research because it is efficient and broken away from the various shortcoming of traditional vaccine, has wide applicating and exploitation prospect.Outer membrane protein (outer membrane protein; OM protein) is one of the main component of gram negative bacteria adventitia, physiological activities such as because outer membrane protein is directly in the face of host's body fluid and tissue, antibacterial utilizes that outer membrane protein sticks, intrusion and inflammation; Discerned as target of attack by the host more easily; Make the outer membrane protein of gram negative bacteria have good immunogenicity, not only can stimulate humoral immunization, and the pair cell immunity also has stimulation.In recent years; Some outer membrane protein of antibacterial have been proved has good immunogenicity; Pair cell immunity and humoral immunization all have stimulation, have good prospect with its preparation recombinant vaccine, for example in escherichia coli, find the immunity protection function of outer membrane protein OmpX.Yet the immunity protection function of vibrio parahaemolyticus outer membrane protein VP2850 and application thereof are not in the news as yet.
Summary of the invention
The invention provides the method for preparing of vibrio parahaemolyticus outer membrane protein VP2850, may further comprise the steps:
1) amplification of vibrio parahaemolyticus outer membrane protein VP2850 gene;
2) clone of vibrio parahaemolyticus outer membrane protein VP2850 gene, screening;
3) vibrio parahaemolyticus outer membrane protein VP2850 expression of gene;
4) purification of vibrio parahaemolyticus outer membrane protein VP2850.
The present invention also provides the application of vibrio parahaemolyticus outer membrane protein VP2850 in the vaccine component that the anti-vibrio parahaemolytious of preparation, vibrio alginolyticus, Pseudomonas fluorescence and Aeromonas hydrophila infect.
The passive immunity protection test of vibrio parahaemolyticus outer membrane protein VP2850, active immunity protection test and active cross immunity protection test result show: can significantly improve the resistance power of Carassius auratus to pathogenic vibrio parahaemolytious behind the vibrio parahaemolyticus outer membrane protein VP2850 immunity Carassius auratus; Can also improve Carassius auratus and mice resistance behind immunity Carassius auratus and the mice to vibrio alginolyticus, Pseudomonas fluorescence and Aeromonas hydrophila.This shows; Vibrio parahaemolyticus outer membrane protein VP2850 has tangible immanoprotection action; Can be used as vaccine component or bacterial-infection resisting preparation, be used to prevent and treat vibrio parahaemolytious, vibrio alginolyticus, Pseudomonas fluorescence and Aeromonas hydrophila and infect the disease that is caused.
Description of drawings
Fig. 1 is a VP2850 gene PCR product agarose gel electrophoresis analysis chart, and wherein M is a dna molecular amount standard;
Fig. 2 is a VP1192 gene PCR product agarose gel electrophoresis analysis chart, and wherein M is a dna molecular amount standard;
Fig. 3 identifies figure for reorganization PV2850 Gene Double enzyme action, and wherein M is a dna molecular amount standard;
Fig. 4 identifies figure for reorganization PV1192 Gene Double enzyme action, and wherein M is a dna molecular amount standard;
Fig. 5 is that the PAGE of VP2850 gene expression and purified product detects figure, and wherein a is a gene expression product, and b is a purified product;
Fig. 6 is that the PAGE of VP1192 gene expression and purified product detects figure, and wherein a is a gene expression product, and b is a purified product;
Fig. 7 is the MALDI-TOF/MS peptide finger printing of vibrio parahaemolyticus outer membrane protein VP2850.
The specific 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.
Embodiment one: the preparation of vibrio parahaemolyticus outer membrane protein VP2850 and purification
Gene coded sequence or the fragment of the vibrio parahaemolyticus outer membrane protein VP2850 of total length are built in the Escherichia coli protein prokaryotic expression carrier, to express and the purification destination protein.
Vibrio parahaemolyticus outer membrane protein VP2850 gene coded sequence is following:
ttatttagcg?aagttgatga?tagggaaaca?tttgaagaag?ccgttatccg 50
cacagttcaa?cagaccaatt?tgcacgccat?cgatttggtc?agtcatgttg 100
aagaagccaa?gttggaagtt?agacttttta?gaaatactcg?ccagaccaac 150
atctgccatg?gtgtaacctt?ctgagtagtt?caccgcactc?cagtttaggc 200
ctttcacgtt?gttcgtgatg?tttaccgcac?caaagttcac?accagttgtt 250
tggccttggt?tccagttaac?caaaccaagt?gacgcacctt?tcatctcttg 300
gtttactttc?gctgccccaa?agaacagacc?gaagttcaca?cccgtagtac 350
ggtcagtttc?agacatacct?agaactgaga?agtcgacgcc?ttttacttcg 400
tttacttggc?catgcagtac?cgccaaacga?acaccaccaa?cagcagagtt 450
tgatggagcg?ttagtatggt?cgatcgttga?aaacatcaca?ggggtgctgt 500
tcgcaagagc?aacaggtgat?gcgatagtgg?ctgcaacagc?caatgacgtc 550
ataagctttt?tcatttttat?ctccat 576
Vibrio parahaemolyticus outer membrane protein VP2850 aminoacid sequence is following:
MEIKMKKLMT?SLAVAATIAS?PVALANSTPV?MFSTIDHTNA?PSNSAVGGVR 50
LAVLHGQVNE?VKGVDFSVLG?MSETDRTTGV?NFGLFFGAAK?VNQEMKGASL 100
GLVNWNQGQT?TGVNFGAVNI?TNNVKGLNWS?AVNYSEGYTM?ADVGLASISK 150
KSNFQLGFFN?MTDQIDGVQI?GLLNCADNGF?FKCFPIINFA?K 191
1, the amplification of vibrio parahaemolyticus outer membrane protein VP2850 gene
The whole genome sequence of the vibrio parahaemolytious of announcing according to NCBI designs a pair of primer, and primer sequence is following:
Forward primer (Sense primer): 5 '-GCGGAATTCTTACTGCTTACCATCAT-3 ';
Reverse primer (Anti-sense Primer): 5 '-TGTCTCGAGTTTGGTATCTGGCTTT-3 '.
For ease of the structure of clone and expression vector, on primer, introduce restriction endonuclease sites EcoR I and Xho I respectively.Primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
The full genome of the vibrio parahaemolytious that obtains with the thermal denaturation method is a template; Carry out the PCR reaction; Reaction system is following: PCR reaction buffer 2.5 μ l, and 10mmol/LdNTP is totally 1 μ l, each 1 μ l of 10 μ mol/L forward primers and reverse primer; Template DNA 1～2 μ l is supplemented to 25 μ l with ultra-pure water with reaction system.PCR reaction cycle parameter is following: the first step: 94 ℃ of 2min; Second step: 94 ℃ of degeneration 60s, 52 ℃ of annealing 60s, 72 ℃ are extended 60s, 30 circulations.Extend 10min with continued at 72 ℃, and preserve down at 4 ℃.Come the segmental length (see figure 1) of identification of dna and reclaim dna fragmentation with agarose gel electrophoresis.
2, the clone of vibrio parahaemolyticus outer membrane protein VP2850 gene, screening and evaluation
With restricted enzyme EcoR I and Xho I difference enzyme action dna fragmentation and pET-32a (Novagen) expression vector; Dna ligation kit operation instructions with reference to Takara company carry out coupled reaction; Reaction system is following: enzymatic solution 5 μ l, through the PCR of enzyme action product 1 μ l, through the pET-32a of enzyme action expression vector 0.5 μ l; With ultra-pure water reaction system is supplemented to 10 μ l, in about 16 ℃, is incubated 14～16 hours.With recombinant products transformed into escherichia coli DH5 α competence, after the conversion thalline is applied on the LB solid plate that contains ampicillin (50 μ g/mL), cultivated 12～16 hours in 37 ℃.Picking contains the recon on the LB flat board of ampicillin at random; Be inoculated on the LB fluid medium that contains ampicillin (50 μ g/mL); Inoculation simultaneously contains the bacillus coli DH 5 alpha that free pET-32a plasmid is arranged;, extract plasmid respectively with alkaline lysis and carry out double digestion evaluation (see figure 3) after 12～16 hours in 37 ℃ of cultivations.To have the recon of insertion to deliver to Shanghai Sangon Biological Engineering Technology And Service Co., Ltd and carry out sequencing, the result show with NCBI in the vibrio parahaemolyticus outer membrane protein VP2850 that reports 98% homology is arranged, explain to have obtained correct reorganization PV2850 gene.
3, vibrio parahaemolyticus outer membrane protein VP2850 expression of gene
Correct reorganization PV2850 gene is expressed bacterium with heat shock method transformed into escherichia coli BL21, cultivated 12～16 hours in 37 ℃.With single colony inoculation in the LB fluid medium that contains ampicillin (50 μ g/mL); Inoculation simultaneously contain free pET-32a plasmid is arranged e. coli bl21 as contrast, being cultured to OD value in 37 ℃ is 0.6, adding 0.5mmol/L IPTG; Induced centrifugal collection thalline 3 hours in 35 ℃.Detect the whether molecular weight size of expressing protein and expressing protein of reorganization PV2850 gene through SDS-PAGE, the result is as shown in Figure 5.
4, the purification of vibrio parahaemolyticus outer membrane protein VP2850
Large scale cultivating transforms thalline, carries out supersound process after centrifugal, and centrifugal collection supernatant carries out purification with Ni-NTA His Bind affinity column.The first step is with dcq buffer liquid Buffer E (8M carbamide, 0.1M NaH
2PO
4, 10mm Tris-HCl, regulate pH value to 6.3 with HCl) washing 5 times, each consumption is 1ml; Second step is with elution buffer Buffer F (8M carbamide, 0.1M NaH
2PO
4, 10mm Tris-HCl, regulate pH value to 5.9 with HCl) eluting destination protein 4 times, each consumption is 1ml, collects eluent A; The 3rd step is with elution buffer Buffer G (8M carbamide, 0.1M NaH
2PO
4, 10mmTris-HCl, regulate pH value to 4.5 with HCl) eluting do not combine the albumen 4 times of Ni-NTA His Bind affinity column, each consumption is 1ml, collects eluent B.The eluent A and the eluent B that get collection carry out the SDS-PAGE analysis, and purification result is as shown in Figure 5.Can know that by Fig. 5 the proteic molecular weight of reorganization PV2850 gene expression all is equivalent to the proteic molecular weight of wild type VP2850 gene expression, promptly than the little 20kD of reality, points out it not contain the fusion protein molecule amount.For proving this inference, through mass spectrum the purifying protein of recombinant bacterial strain high expressed is analyzed, mass spectrum result (as shown in Figure 7) shows that this albumen is vibrio parahaemolyticus outer membrane protein VP2850 (seeing table 1).The vibrio parahaemolyticus outer membrane protein VP2850 packing that purification is good, behind the lyophilization powdered, frozen in-80 ℃ of refrigerators.
The Matrix Mowse result for retrieval of table 1 vibrio parahaemolyticus outer membrane protein VP2850 peptide quality fingerprinting collection of illustrative plates
Embodiment two: the preparation of vibrio parahaemolyticus outer membrane protein VP1192 and purification
Gene coded sequence or the fragment of the vibrio parahaemolyticus outer membrane protein VP1192 of total length are built in the Escherichia coli protein prokaryotic expression carrier, to express and the purification destination protein.
Vibrio parahaemolyticus outer membrane protein VP1192 gene coded sequence is following:
ttagcgaggt?acaacccttg?cgtcattacc?agacatgttg?atttgaacgg 50
cttggcctgg?ctggaaaatc?atgtttgggt?ttgcttcttg?aacgacagat 100
atgatctttc?catcctctaa?gcgaatagtg?aggttcacac?catttctttt 150
cgcggttgct?tcggcggctt?tactacctgc?atagccacct?aataaaccac 200
caccaatggc?tgcgatatcc?gagccagaac?cgcccccaat?tttcgaaccc 250
aaaatgccac?ccacggcagc?ccctgctatt?gtaccgatgg?cgttggattg 300
ggtagaggca?tcaatggtta?cgggttctac?tttttctatc?gtgccgtagt 350
aaacctgctg?aactttgcga?gtgtcggacg?agccgtatgc?atctccgtac 400
gggttaggtg?agctacagcc?gccaagcacg?atcgcacaag?taatggtgag 450
caacatgcct?aacttgatgt gttttttcat ?480
Vibrio parahaemolyticus outer membrane protein VP1192 aminoacid sequence is following:
MKKHIKLGML?LTITCAIVLG?GCSSPNPYGD?AYGSSDTRKV?QQVYYGTIEK 50
VEPVTIDAST?QSNAIGTIAG?AAVGGILGSK?IGGGSGSDIA?AIGGGLLGGY 100
AGSKAAEATA?KRNGVNLTIR?LEDGKIISVV?QEANPNMIFQ?PGQAVQINMS 150
GNDARVVPR 159
1, the amplification of vibrio parahaemolyticus outer membrane protein VP1192 gene
The whole genome sequence of the vibrio parahaemolytious of announcing according to NCBI designs a pair of primer, and primer sequence is following:
Forward primer (Sense primer): 5 ' CGC
GGATCCATGAAAAAACACATCAAGT-3 ';
Reverse primer (Anti-sense Primer): 5 '-AGC
AAGCTTTTAGCGAGGTACAACCC-3 '.
For ease of the structure of clone and expression vector, on primer, introduce restriction endonuclease sites BamHI and HindIII respectively.Primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
The full genome of the vibrio parahaemolytious that obtains with the thermal denaturation method is a template; Carry out the PCR reaction; Reaction system is following: PCR reaction buffer 2.5 μ l, and 10mmol/L dNTP is totally 1 μ l, each 1 μ l of 10 μ mol/L forward primers and reverse primer; Template DNA 1～2 μ l is supplemented to 25 μ l with ultra-pure water with reaction system.PCR reaction cycle parameter is following: the first step: 94 ℃ of 2min; Second step: 94 ℃ of degeneration 60s, 60 ℃ of annealing 60s, 72 ℃ are extended 60s, 30 circulations.Extend 10min with continued at 72 ℃, and preserve down at 4 ℃.Come the segmental length (see figure 2) of identification of dna and reclaim dna fragmentation with agarose gel electrophoresis.
2, the clone of vibrio parahaemolyticus outer membrane protein VP1192 gene, screening and evaluation
With restricted enzyme BamHI and Hind III difference enzyme action dna fragmentation and pET-32a (Novagen) expression vector; Dna ligation kit operation instructions with reference to Takara company carry out coupled reaction; Reaction system is following: enzymatic solution 5 μ l, through the PCR of enzyme action product 1 μ l, through the pET-32a of enzyme action expression vector 0.5 μ l; With ultra-pure water reaction system is supplemented to 10 μ l, in about 16 ℃, is incubated 14～16 hours.With recombinant products transformed into escherichia coli DH5 α competence, after the conversion thalline is applied on the LB solid plate that contains ampicillin (50 μ g/mL), cultivated 12～16 hours in 37 ℃.Picking contains the recon on the LB flat board of ampicillin at random; Be inoculated on the LB fluid medium that contains ampicillin (50 μ g/mL); Inoculation simultaneously contains the bacillus coli DH 5 alpha that free pET-32a plasmid is arranged;, extract plasmid respectively with alkaline lysis and carry out double digestion evaluation (see figure 4) after 12～16 hours in 37 ℃ of cultivations.To have the recon of insertion to deliver to Shanghai Sangon Biological Engineering Technology And Service Co., Ltd and carry out sequencing, the result show with NCBI in the vibrio parahaemolyticus outer membrane protein 1192 reported 97.5% homology is arranged, explain to have obtained correct reorganization PV1192 gene.
3, vibrio parahaemolyticus outer membrane protein VP1192 expression of gene
Correct reorganization PV1192 gene is expressed bacterium with heat shock method transformed into escherichia coli BL21, cultivated 12～16 hours in 37 ℃.With single colony inoculation in the LB fluid medium that contains ampicillin (50 μ g/mL); Inoculation simultaneously contain free pET-32a plasmid is arranged e. coli bl21 as contrast, being cultured to OD value in 37 ℃ is 0.6, adding 0.5mmol/L IPTG; Induced centrifugal collection thalline 3 hours in 35 ℃.Detect the whether molecular weight size of expressing protein and expressing protein of reorganization PV1192 gene through SDS-PAGE, the result is as shown in Figure 6.
4, the purification of vibrio parahaemolyticus outer membrane protein VP1192
Large scale cultivating transforms thalline, carries out supersound process after centrifugal, and centrifugal collection supernatant carries out purification with Ni-NTA His Bind affinity column.The first step is with dcq buffer liquid Buffer E (8M carbamide, 0.1M NaH
2PO
4, 10mm Tris-HCl, regulate pH value to 6.3 with HCl) washing 5 times, each consumption is 1ml; Second step is with elution buffer Buffer F (8M carbamide, 0.1M NaH
2PO
4, 10mm Tris-HCl, regulate pH value to 5.9 with HCl) eluting destination protein 4 times, each consumption is 1ml, collects eluent A; The 3rd step is with elution buffer Buffer G (8M carbamide, 0.1M NaH
2PO
4, 10mmTris-HCl, regulate pH value to 4.5 with HCl) eluting do not combine the albumen 4 times of Ni-NTA His Bind affinity column, each consumption is 1ml, collects eluent B.The eluent A and the eluent B that get collection carry out the SDS-PAGE analysis, and purification result is as shown in Figure 6.The vibrio parahaemolyticus outer membrane protein VP1192 packing that purification is good, behind the lyophilization powdered, frozen in-80 ℃ of refrigerators.
Embodiment three: the sero-fast passive immunity protective effect of vibrio parahaemolyticus outer membrane protein VP2850
Vibrio parahaemolyticus outer membrane protein VP2850 immunizing rabbit with embodiment one prepared purification obtains the VP2850 antiserum.Concrete steps are following: vibrio parahaemolyticus outer membrane protein VP2850 lyophilized powder is dissolved in an amount of ultra-pure water; Then with isopyknic Freund's complete adjuvant (lanoline: liquid paraffin=1: 4v/v; Bacillus calmette-guerin vaccine is 30mg/ml) mix homogeneously, wherein protein content is about 500 μ g, supersound process 2min then; Promptly make VP2850 antigen, adopt subcutaneous multi-point injection; After 20 days, (lanoline: liquid paraffin=1: 4v/v) mix, inject to rabbit, wherein protein content is about 500 μ g with isopyknic incomplete Freund with vibrio parahaemolyticus outer membrane protein VP2850; After 20 days again injection once, rear neck artery was got blood in 7 days, 4 ℃ separate out serum after spending the night naturally; 4 ℃ 3000 rev/mins, centrifugal 30 minutes, get supernatant and promptly get the VP2850 antiserum.
By the same way, the vibrio parahaemolyticus outer membrane protein VP1192 immunizing rabbit with embodiment two prepared purification obtains the VP1192 antiserum, uses antiserum as controlled trial.
Adopt the Dot-ELISA technology, measure respectively that VP2850 antiserum and VP1192 are sero-fast to tire.Recording sero-fast the tiring of VP2850 is 2.5 ten thousand, and sero-fast the tiring of VP1192 is 50,000.
The Carassius auratus of buying (being about the 20g/ tail) was raised for 1 week, and water temperature is controlled at 25 ± 2 ℃, and every day is twice artifical compound feed of throwing something and feeding sooner or later, raises and train the N/R random packet in 1 week back and carries out inoculation.According to the result of above-mentioned Dot-ELISA, get and equate that the VP2850 antiserum and the VP1192 antiserum of tiring carry out passive immunity to Carassius auratus (about 20 gram/bars) respectively.In the experimental group, give Carassius auratus lumbar injection 100 μ l antiserums, count the vibrio parahaemolytious (5 * 10 of fatal dose after 2.5 hours with 5 sesquialters
8Cfu/ml) infect.In the blank group, give Carassius auratus lumbar injection 100 μ l normal saline after, infect with the vibrio parahaemolytious of equivalent.Observe the existence situation of respectively organizing Carassius auratus after 72 hours, calculate the relative immunity protective rate of respectively organizing Carassius auratus.The result shows that the sero-fast relative immunity protective rate of VP2850 is 75.0%, and the sero-fast relative immunity protective rate of VP1192 is merely 15.0%, the Carassius auratus of blank group all dead (seeing table 2).Three groups of data have the difference of highly significant each other, show that vibrio parahaemolyticus outer membrane protein VP2850 has tangible passive immunity protective effect.
Relative immunity protective rate (%)=(1-test group mortality rate/matched group mortality rate) * 100%
The passive immunity protective effect of table 2 vibrio parahaemolyticus outer membrane protein VP2850
*P＜0.05
Embodiment four: the active immunity protective effect of vibrio parahaemolyticus outer membrane protein VP2850
Raise Carassius auratus according to embodiment three described conditions, raise and train the N/R random packet in 1 week back and carry out inoculation.The vibrio parahaemolyticus outer membrane protein VP2850 lyophilized powder of embodiment one prepared purification is dissolved in an amount of ultra-pure water; Then with isopyknic Freund's complete adjuvant (lanoline: liquid paraffin=1: 4v/v; Bacillus calmette-guerin vaccine is 30mg/ml) mix homogeneously; Wherein protein content is about 25 μ g, and supersound process 2min promptly makes VP2850 antigen then.In the experimental group, give Carassius auratus lumbar injection VP2850 antigen, 25 μ g/ tail fishes; After 10 days, with vibrio parahaemolyticus outer membrane protein VP2850 and isopyknic incomplete Freund (lanoline: liquid paraffin=1: 4v/v) mix, carry out lumbar injection, 12.5 μ g/ tail fishes to Carassius auratus.In the blank group, give Carassius auratus intraperitoneal injection of saline 100 μ l, whenever, inject altogether 2 times at a distance from injection in 10 days 1 time.
The 2nd inoculation organized Carassius auratus is counted fatal dose with 5 sesquialters vibrio parahaemolytious (5 * 10 after 7 days to each
8Cfu/mL) carry out the experiment of counteracting toxic substances protectiveness.Observe the existence situation of respectively organizing Carassius auratus after 72 hours, calculate the relative immunity protective rate respectively organize Carassius auratus and the protection effect of comparative analysis immunogenic protein in view of the above.The result shows that the relative immunity protective rate of vibrio parahaemolyticus outer membrane protein VP2850 immunity Carassius auratus antagonism vibrio parahaemolytious counteracting toxic substances is 83.3% (seeing table 3).The result shows that vibrio parahaemolyticus outer membrane protein VP2850 has tangible active immunity protective effect.
The active immunity protective effect of table 3 vibrio parahaemolyticus outer membrane protein VP2850
*P＜0.01
Embodiment five: the protective effect of the sero-fast active cross immunity of vibrio parahaemolyticus outer membrane protein VP2850
1, to the active cross immunity protective effect of Carassius auratus
Make an experiment with reference to embodiment four described methods, wherein carrying out the used antibacterial of counteracting toxic substances protectiveness experiment is vibrio alginolyticus, Pseudomonas fluorescence and Aeromonas hydrophila.Counteracting toxic substances dosage is decided to be: vibrio alginolyticus 5 * 10
8Cfu/ml, Pseudomonas fluorescence 5 * 10
8Cfu/ml, Aeromonas hydrophila 4 * 10
8Cfu/ml.Observe the existence situation of respectively organizing Carassius auratus after 120 hours, calculate the relative immunity protective rate of respectively organizing Carassius auratus.The result shows that the relative immunity protective rate of vibrio parahaemolyticus outer membrane protein VP2850 antagonism vibrio alginolyticus, Pseudomonas fluorescence and Aeromonas hydrophila counteracting toxic substances is followed successively by 83.3%, 92.9% and 66.7% (seeing table 4).
Table 4 vibrio parahaemolyticus outer membrane protein VP2850 is to the active cross immunity protective effect of Carassius auratus
*P＜0.05
2, to the active cross immunity protective effect of mice
Adopt Kunming mouse about about 3 weeks as test mice, carry out the experiment of counteracting toxic substances protectiveness with vibrio alginolyticus, Pseudomonas fluorescence and Aeromonas hydrophila respectively.
At first mice is carried out the immunity of vibrio parahaemolyticus outer membrane protein VP2850.Immunologic process is following: the vibrio parahaemolyticus outer membrane protein VP2850 lyophilized powder of embodiment one prepared purification is dissolved in an amount of ultra-pure water; Then with isopyknic Freund's complete adjuvant (lanoline: liquid paraffin=1: 4v/v; Bacillus calmette-guerin vaccine is 30mg/ml) mix homogeneously; Wherein protein content is about 100 μ g, and supersound process 2min promptly makes VP2850 antigen then.In the experimental group, give Kunming mouse lumbar injection VP2850 antigen; After 10 days, (lanoline: liquid paraffin=1: 4v/v) mix, wherein protein content is about 100 μ g, carries out lumbar injection to Kunming mouse with isopyknic incomplete Freund with vibrio parahaemolyticus outer membrane protein VP2850.In the blank group, give Kunming mouse intraperitoneal injection of saline 100 μ l, inject again after 10 days 1 time.
The 2nd inoculation carried out the experiment of counteracting toxic substances protectiveness to mice after about 7 days.Counteracting toxic substances dosage is decided to be: vibrio alginolyticus 5 * 10
9Cfu/ml, Pseudomonas fluorescence 5 * 10
9Cfu/ml, Aeromonas hydrophila 4 * 10
9Cfu/ml.Observe the existence situation of respectively organizing mice after 72 hours, calculate the relative immunity protective rate of respectively organizing mice.The result shows that the relative immunity protective rate of vibrio parahaemolyticus outer membrane protein VP2850 antagonism vibrio alginolyticus, Pseudomonas fluorescence and Aeromonas hydrophila counteracting toxic substances is followed successively by 80.0%, 68.4% and 73.3% (seeing table 5).
Table 5 vibrio parahaemolyticus outer membrane protein VP2850 is to the active cross immunity protective effect of mice
*P＜0.05；
**P＜0.01
The result shows that vibrio parahaemolyticus outer membrane protein VP2850 has the protective effect of tangible active cross immunity.
In sum; Vibrio parahaemolyticus outer membrane protein VP2850 has tangible immanoprotection action; Can be used as vaccine component or bacterial-infection resisting preparation, be used to prevent and treat vibrio parahaemolytious, vibrio alginolyticus, Pseudomonas fluorescence and Aeromonas hydrophila and infect the disease that is caused.
Sequence table
< 110>Zhongshan University
< 120>application of the preparation of vibrio parahaemolyticus outer membrane protein VP2850 and immunity protection function thereof
<160>4
<210>1
<211>576
<212>DNA
< 213>vibrio parahaemolyticus outer membrane protein VP2850 encoding gene
<400>1
ttatttagcg?aagttgatga?tagggaaaca?tttgaagaag?ccgttatccg 50
cacagttcaa?cagaccaatt?tgcacgccat?cgatttggtc?agtcatgttg 100
aagaagccaa?gttggaagtt?agacttttta?gaaatactcg?ccagaccaac 150
atctgccatg?gtgtaacctt?ctgagtagtt?caccgcactc?cagtttaggc 200
ctttcacgtt?gttcgtgatg?tttaccgcac?caaagttcac?accagttgtt 250
tggccttggt?tccagttaac?caaaccaagt?gacgcacctt?tcatctcttg 300
gtttactttc?gctgccccaa?agaacagacc?gaagttcaca?cccgtagtac 350
ggtcagtttc?agacatacct?agaactgaga?agtcgacgcc?ttttacttcg 400
tttacttggc?catgcagtac?cgccaaacga?acaccaccaa?cagcagagtt 450
tgatggagcg?ttagtatggt?cgatcgttga?aaacatcaca?ggggtgctgt 500
tcgcaagagc?aacaggtgat?gcgatagtgg?ctgcaacagc?caatgacgtc 550
ataagctttt?tcatttttat?ctccat 576
<210>2
<211>191
<212>PRT
< 213>vibrio parahaemolyticus outer membrane protein VP2850
<400>2
Met?Glu?Ile?Lys?Met?Lys?Lys?Leu?Met?Thr?Ser?Leu?Ala?Val?Ala
5 10 15
Ala?Thr?Ile?Ala?Ser?Pro?Val?Ala?Leu?Ala?Asn?Ser?Thr?Pro?Val
20 25 30
Met?Phe?Ser?Thr?Ile?Asp?His?Thr?Asn?Ala?Pro?Ser?Asn?Ser?Ala
35 40 45
Val?Gly?Gly?Val?Arg?Leu?Ala?Val?Leu?His?Gly?Gln?Val?Asn?Glu
50 55 60
Val?Lys?Gly?Val?Asp?Phe?Ser?Val?Leu?Gly?Met?Ser?Glu?Thr?Asp
65 70 75
Arg?Thr?Thr?Gly?Val?Asn?Phe?Gly?Leu?Phe?Phe?Gly?Ala?Ala?Lys
80 85 90
Val?Asn?Gln?Glu?Met?Lys?Gly?Ala?Ser?Leu?Gly?Leu?Val?Asn?Trp
95 100 105
Asn?Gln?Gly?Gln?Thr?Thr?Gly?Val?Asn?Phe?Gly?Ala?Val?Asn?Ile
110 115 120
Thr?Asn?Asn?Val?Lys?Gly?Leu?Asn?Trp?Ser?Ala?Val?Asn?Tyr?Ser
125 130 135
Glu?Gly?Tyr?Thr?Met?Ala?Asp?Val?Gly?Leu?Ala?Ser?Ile?Ser?Lys
140 145 150
Lys?Ser?Asn?Phe?Gln?Leu?Gly?Phe?Phe?Asn?Met?Thr?Asp?Gln?Ile
155 160 165
Asp?Gly?Val?Gln?Ile?Gly?Leu?Leu?Asn?Cys?Ala?Asp?Asn?Gly?Phe
170 175 180
Phe?Lys?Cys?Phe?Pro?Ile?Ile?Asn?Phe?Ala?Lys
185 190
<210>3
<211>480
<212>DNA
< 213>vibrio parahaemolyticus outer membrane protein VP1192 encoding gene
<400>3
ttagcgaggt?acaacccttg?cgtcattacc?agacatgttg?atttgaacgg 50
cttggcctgg?ctggaaaatc?atgtttgggt?ttgcttcttg?aacgacagat 100
atgatctttc?catcctctaa?gcgaatagtg?aggttcacac?catttctttt 150
cgcggttgct?tcggcggctt?tactacctgc?atagccacct?aataaaccac 200
caccaatggc?tgcgatatcc?gagccagaac?cgcccccaat?tttcgaaccc 250
aaaatgccac?ccacggcagc?ccctgctatt?gtaccgatgg?cgttggattg 300
ggtagaggca?tcaatggtta?cgggttctac?tttttctatc?gtgccgtagt 350
aaacctgctg?aactttgcga?gtgtcggacg?agccgtatgc?atctccgtac 400
gggttaggtg?agctacagcc?gccaagcacg?atcgcacaag?taatggtgag 450
caacatgcct?aacttgatgt?gttttttcat 480
<210>4
<211>159
<212>PRT
< 213>vibrio parahaemolyticus outer membrane protein VP1192
<400>4
Met?Lys?Lys?His?Ile?Lys?Leu?Gly?Met?Leu?Leu?Thr?Ile?Thr?Cys
5 10 15
Ala?Ile?Val?Leu?Gly?Gly?Cys?Ser?Ser?Pro?Asn?Pro?Tyr?Gly?Asp
20 25 30
Ala?Tyr?Gly?Ser?Ser?Asp?Thr?Arg?Lys?Val?Gln?Gln?Val?Tyr?Tyr
35 40 45
Gly?Thr?Ile?Glu?Lys?Val?Glu?Pro?Val?Thr?Ile?Asp?Ala?Ser?Thr
50 55 60
Gln?Ser?Asn?Ala?Ile?Gly?Thr?Ile?Ala?Gly?Ala?Ala?Val?Gly?Gly
65 70 75
Ile?Leu?Gly?Ser?Lys?Ile?Gly?Gly?Gly?Ser?Gly?Ser?Asp?Ile?Ala
80 85 90
Ala?Ile?Gly?Gly?Gly?Leu?Leu?Gly?Gly?Tyr?Ala?Gly?Ser?Lys?Ala
95 100 105
Ala?Glu?Ala?Thr?Ala?Lys?Arg?Asn?Gly?Val?Asn?Leu?Thr?Ile?Arg
110 115 120
Leu?Glu?Asp?Gly?Lys?Ile?Ile?Ser?Val?Val?Gln?Glu?Ala?Asn?Pro
125 130 135
Asn?Met?Ile?Phe?Gln?Pro?Gly?Gln?Ala?Val?Gln?Ile?Asn?Met?Ser
140 145 150
Gly?Asn?Asp?Ala?Arg?Val?Val?Pro?Arg
155
Claims (1)
1. the application of vibrio parahaemolyticus outer membrane protein VP2850 in the vaccine component that the anti-vibrio parahaemolytious of preparation, vibrio alginolyticus, Pseudomonas fluorescence and Aeromonas hydrophila infect.
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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 |
| CN101020051A (en) * | 2006-12-22 | 2007-08-22 | 广东海洋大学 | Prepn and usage of outer membrane protein subunit vaccine of seawater fish morbid vibrio |
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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 |
| CN101020051A (en) * | 2006-12-22 | 2007-08-22 | 广东海洋大学 | Prepn and usage of outer membrane protein subunit vaccine of seawater fish morbid vibrio |
| CN101172157A (en) * | 2007-09-21 | 2008-05-07 | 浙江大学 | Vibrio parahaemolyticus tunica externa protein ompK subunit vaccine and preparation method thereof |
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