CN108330142A - A kind of Mermaid luminous bacillus hemolysin Hly with immanoprotection actionchAlbumen - Google Patents

A kind of Mermaid luminous bacillus hemolysin Hly with immanoprotection actionchAlbumen Download PDF

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CN108330142A
CN108330142A CN201810132388.9A CN201810132388A CN108330142A CN 108330142 A CN108330142 A CN 108330142A CN 201810132388 A CN201810132388 A CN 201810132388A CN 108330142 A CN108330142 A CN 108330142A
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hly
albumen
luminous bacillus
mermaid luminous
hemolysin
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吴同垒
高桂生
张志强
史秋梅
肖丽荣
李巧玲
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Hebei Normal University of Science and Technology
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Abstract

The invention discloses a kind of Mermaid luminous bacillus hemolysin Hly with immanoprotection actionchAlbumen, the present invention is by building recombinant vector pMD18T hlych, recombinant expression carrier pET32a hlychWith prokaryotic expression hlychGene, Prepare restructuring HlychAlbumen, and verify Hly using SDS PAGE analyses and Western blotchAlbumen and animal protection experiment evaluate its protection.The Hly of the Mermaid luminous bacillus obtained in the present inventionchAlbumen has good immune protective efficiency.It lays a good foundation for exploitation Mermaid luminous bacillus engineering subunit vaccine.

Description

A kind of Mermaid luminous bacillus hemolysin Hly with immanoprotection actionchAlbumen
Technical field
The present invention relates to biotechnologies, more particularly to Mermaid luminous bacillus hemolysin HlychAlbumen Preparation and identification method.
Background technology
Mermaid luminous bacillus (Photobacterium damselae) is the important member in thermophilic salt pathogen, infection For the illness fish clinic of the bacterium using hueppe's disease as characteristic feature, morbidity is rapid, and the death rate is high, to marine economy fish culture Industry causes heavy economic losses.People and other mammals can also infect, with a kind of pathogenic bacteria that people-beast-fish suffers from altogether. The Mermaid luminous bacillus of known highly pathogenicity usually carries the virulence plasmid of about 150kb, two kinds of critical virulence of coding because Son-hemolysin Dly albumen and HlyAplAlbumen, while hemolysin Hly is also carried in chromatinchGene, these three hemolysins are all There is different degrees of influence to the virulence of bacterium.Hemolysin is important one of the virulence factor of Mermaid luminous bacillus, Neng Gouzuo For the lipid bilayer of red blood cell, cell membrane is made to form cavity, changes membrane passage, cause the rupture of red blood cell Dissolving, to release more ferrohemes, more Fe are provided for it3+, eventually lead to marine cultured animal histoorgan Necrosis, or even cause the death of infection animal.
The turbot (Turbot) in November, 2015, the cultivation of Qinhuangdao Changli County sea-farming factory is largely fallen ill, sick fish Show as body surface ulcer, fin bleeding, tail portion are festered, serious ascites, lethality is up to 80%, highlights important aetology meaning Justice.This seminar detaches from ascites and identifies Mermaid luminous bacillus, and is named as by Chinese Sea Organism Depositary MCCC 1K03226.The important function of highly pathogenicity and hemolysin in view of Mermaid luminous bacillus, research are Mermaid luminous The Carriage of bacillus hemolysin and the immune protective effect of hemolysin finally reach to develop genetic engineering subunit vaccine To the purpose for controlling the disease, the problem of being those skilled in the art's urgent need to resolve.
Invention content
In view of this, the present invention provides a kind of Mermaid luminous bacillus hemolysin Hly with immanoprotection actionchEgg In vain, the present invention is expressed and is purified to it using prokaryotic expression system, analysis hemolysin HlychThe immunogenicity of albumen, action of going forward side by side Object Protection.To achieve the goals above, the present invention adopts the following technical scheme that：
A kind of Mermaid luminous bacillus hemolysin Hly with immanoprotection actionchAlbumen is developing Mermaid luminous bar It is applied in bacterium genetic engineering subunit vaccine.
Further, a kind of Mermaid luminous bacillus hemolysin Hly with immanoprotection actionchThe system of albumen Preparation Method is as follows
1) recombinant vector pMD18T-hlychStructure
The genomic DNA of Mermaid luminous bacillus is extracted, the culture presevation number of the Mermaid luminous bacillus is MCCC1K03226, using genomic DNA as template, with Hlych-F、Hlych- R is that primer amplification removes signal coding sequence HlychGene expands Hly using touchdown PCRchFull length gene, the primer Hlych-F、HlychThe sequence of-R is as follows：
Hlych-F：5'-GCGGATCCGACGTTATATCAATATTAAGCAG-3'；With I restriction enzyme sites of BamH；SEQ ID NO：10；
Hlych-R：5'-GCGTCGACTTACTGATTTATGGTTGATGGGTC-3'；With Sal I restriction enzyme sites；SEQ ID NO：11；
By the hly of acquisitionchGene is connect with carrier T, obtains recombinant vector pMD18T-hlych, and to the recombination Carrier pMD18T-hlychIt is sequenced, carries out proteins subcellular location；
2) recombinant expression carrier pET32a-hlychStructure
Using restriction enzyme BamH I and Sal I respectively to the recombinant vector pMD18T-hlychAnd expression vector PET32a (+) carries out double digestion, and gel electrophoresis is recycled digestion products, mixed in proportion, and 22-25 DEG C of connection 2-3h converts large intestine In bacillus competent cell, in access LB tablets one, 37 DEG C of overnight incubations；Picking single bacterium colony shakes bacterium, uses carrier universal primer Carry out PCR identifications；Positive bacterium colony is expanded and is cultivated, strain is preserved and extracts plasmid pET32a-hlych；
3)hlychProkaryotic expression
By the plasmid pET32a-hlychIn conversion expression bacterial strain BL21, it is applied in LB tablets two, 37 DEG C were cultivated Night；The single bacterium colony of LB tablets two described in picking is shaken bacterium and is stayed overnight, and next day draws in the LB liquid medium of 1mL bacterium solutions inoculation 100mL, 220r/min shakes bacterium, until OD600IPTG induced expressions 4-6h is added up to being 0.6-0.8 in value.
Further, recombinant vector pMD18T-hly described in step 2)chIt is returned with expression vector pET32a (+) digestion The ratio for receiving product is 3:1.Wherein, the recombinant vector pMD18T-hlychIt is recycled with expression vector pET32a (+) digestion The ratio of product is excessively high or too low, then can lead to recombinant expression carrier pET32a-hlychIt is difficult to build success.
Further, competent cell described in step 2) is DH5 α.
Further, LB tablets described in step 2) one and the LB tablets two are flat for the solid LB containing ampicillin Plate, a concentration of 45-55 μ g/L of the ampicillin.Wherein, the concentration of ampicillin is too low or excessively high, can cause to sieve The positive bacterium colony failure of choosing.
Further, liquid LB described in step 3) is the liquid LB containing ampicillin, the ampicillin A concentration of 45-55 μ g/L.Wherein, the too low then screening pressure of the concentration of ampicillin is inadequate, and recombinant expression carrier is easily lost, Excessively high then bacterial growth is bad.
Further, a concentration of 1mM of IPTG described in step 3).Wherein, the excessive concentration of IPTG, bacterial growth suppression System, too low then expressing quantity are insufficient.
Mermaid luminous bacillus hemolysin HlychThe identification method of albumen, including SDS-PAGE analyses and Western blot Verification；
The bacteria suspension is centrifuged and is collected thalline, PBS is resuspended after washing 3 times, and Loading Buffer are added and boil and split It solves, progress SDS-PAGE, after electrophoresis, result is observed in gel-colored decoloration；It is carried out at the same time Western blot verifying purposes The expression of albumen.
Further, resolving gel concentration is 12% in the SDS-PAGE electrophoresis.Wherein, using the acryloyl of 12% content Amine, Protein Separation effect are preferable.
Further, in the Western blot methods, primary antibody be band His tag monoclonal antibodies, a concentration of 1: 6000, secondary antibody is sheep anti-mouse igg-HRP, a concentration of 1:4000.
The Mermaid luminous bacillus hemolysin HlychAlbumen evaluates its immunogenicity by ELISA；Steps are as follows：
Induce HlychProtein expression, rear using ultrasound cracking thalline, centrifugation takes supernatant precipitation to carry out SDS- respectively PAGE determines HlychAlbumen is expressed for inclusion body；Using QIAGEN Ni-NTA columns to protein purification；It is coated with 10ng/ porin ELISA Plate, mouse immune serum are primary antibody, and HRP- goat-anti rabbit secondary antibodies are enzyme labelled antibody, carry out ELISA measurement, measure antibody effect Valence.
The Mermaid luminous bacillus hemolysin HlychAlbumen assesses its immune protective efficiency by zoopery；
The zoopery appraisal procedure is：After booster immunization, bacterium was carried out every two weeks and attacks poison, intraperitoneal injection Balb/c is small Mouse Mermaid luminous bacillus MCCC 1K03226, dosage 5LD50(i.e. 6 × 107Cfu), the dead mouse of statistics in latter week is infected Quantity.
The present disclosure provides Mermaid luminous bacillus hemolysin HlychThe preparation of albumen and identification method, at least with Lower advantage：
(1) Hly of Mermaid luminous bacilluschAlbumen has good immunogenicity, can be used for establishing the diagnosis sides ELISA Method is laid a good foundation for the quick diagnosis of high virulence Mermaid luminous bacillus disease.
(2) Hly of Mermaid luminous bacillus disclosed by the inventionchIt can make 65% immune mouse after protein immunization mouse It is protected, i.e. the Hly of Mermaid luminous bacillus in the present inventionchAlbumen has wide in Mermaid luminous bacillus vaccine research Wealthy foreground.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technology description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this The embodiment of invention for those of ordinary skill in the art without creative efforts, can also basis The attached drawing of offer obtains other attached drawings.
Fig. 1 attached drawings are Mermaid luminous bacillus MCCC 1K03226 single bacterium colonies provided by the invention on sheep blood plate Hemolytic activity；
Fig. 2 attached drawings are that Mermaid luminous bacillus MCCC 1K03226 single bacterium colonies provided by the invention are molten on rabbit blood plate Blood activity；
Fig. 3 attached drawings are the PCR agarose gel electrophoresis figures of hemolysin gene provided by the invention detection；
Wherein, swimming lane M is Marker；Swimming lane 1 is hlych；Swimming lane 2 is hlypl；Swimming lane 3 is dly；
Fig. 4 attached drawings are hly provided by the inventionchGene PCR agarose gel electrophoresis figure；
Wherein, swimming lane M is Marker；Swimming lane 1 is hlych；
Fig. 5 attached drawings are Hly provided by the inventionchProtein secondary structure prognostic chart；
Fig. 6 attached drawings are hemolysin Hly provided by the inventionchThe SDS-PAGE electrophoresis of albumen；
Wherein, swimming lane 1 is haemolysis fibroin Hlych；Swimming lane 2 is unloaded compares；
Fig. 7 attached drawings are hemolysin Hly provided by the inventionchThe Western blot electrophoretograms of albumen；
Wherein, swimming lane 1 is haemolysis fibroin Hlych；Swimming lane 2 is unloaded compares；
Fig. 8 attached drawings are purifying hemolysin Hly provided by the inventionchThe SDS-PAGE electrophoresis of albumen；
Wherein, swimming lane M is Marker；Swimming lane 1 is hemolysin Hly before purificationchAlbumen；Swimming lane 2 is haemolysis after purification Plain HlychAlbumen；
Fig. 9 attached drawings are that serum Western blot provided by the invention verify analytical electrophoresis figure；
Wherein, swimming lane M is Marker；Swimming lane 1 is positive serum；Swimming lane 2 is negative control.
Figure 10 attached drawings are Hly provided by the inventionchThe mouse immune protective rate of albumen.
Specific implementation mode
The following is a clear and complete description of the technical scheme in the embodiments of the invention, it is clear that described embodiment Only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field The every other embodiment that art personnel are obtained without making creative work belongs to the model that the present invention protects It encloses.
Embodiment 1
Bacterium hemolytic activity is observed
Sheep Blood and rabbit blood plate, picking Mermaid luminous bacillus MCCC 1K03226 single bacterium colonies, in sheep are prepared respectively It crosses on blood and rabbit blood plate, 30 DEG C of cultures for 24 hours, observe its hemolytic activity, as a result as depicted in figs. 1 and 2, wherein as Fig. 1 is Sheep Blood, Fig. 2 are rabbit blood.By Fig. 1 and Fig. 2 it is found that the bacterium is in β type haemolysis, display on Sheep Blood and rabbit blood plate tablet The bacterium has very strong hemolytic activity.
2 recombinant vector pMD18T-hly of embodimentchStructure
102761 (PhddCIP of Photobacterium damselae subsp.Damselae CIP are downloaded from NCBI 102761, gene accession number：NZ_ADBS00000000) whole genome sequence finds dly, hlyAplAnd hlyAchGene order, Design primer, primer sequence are shown in Table 1；Wherein dly gene orders are SEQ ID NO：1；hlyAplGene order is SEQ ID NO： 2；hlyAchGene order is SEQ ID NO：3.Use bacterial genomes extracts kit extraction strain MCCC 1K03226 DNA Genomic DNA, using primer in table 1, expanded using touchdown PCR, used after reaction using genomic DNA as template 1% agarose gel electrophoresis, as a result such as Fig. 3, wherein primer Dly-f inspections, Dly-r inspection amplification Dly Gene Partial length, for examining Survey hemolysin Dly albumen；Primer Hlypl- f inspections, Hlypl- r inspection amplifications HlyplGene Partial length, it is plasmid-encoded for detecting Hemolysin；Primer Hlych- f inspections, Hlych- r inspection amplifications HlychGene Partial length, the hemolysin for detecting chromatin coding.
Table 1：Primer sequence table
As shown in Figure 1, three kinds of hemolysin Dly albumen, HlyplAlbumen and HlychIn albumen, hlychGene is about 420bp inspections It surveys to be positive, therefore judges that the bacterium MCCC 1K03226DNA carry hlychGene, without carrying hlyplWith dly genes.
Further using the genomic DNA of strain MCCC 1K03226 DNA as masterplate, according to the hly of NCBI announcementschGene Sequence carries out signal peptide prediction using Signa IP, utilizes primer Hlych-F、Hlych- R is gone using the method amplification of touchdown PCR Except the hly of signal coding sequencechFull length gene, after reaction with 1% agarose gel electrophoresis, as a result such as Fig. 4.By Fig. 4 It is found that amplified band size meets with expection, about 1812bp.
By hlychFull length gene is connect according to a conventional method with carrier T, obtains recombinant vector pMD18T-hlych, and will recombination Carrier pMD18T-hlychIt is sequenced, sequence analysis is carried out to the workspaces Blastn of sequencing result application NCBI, finds to expand The hly of increasingchGene reaches 99% with Mermaid luminous bacillus hemolysin hlyA genetic homologies, it is thus determined that U.S. has successfully been obtained The hly of mermaid luminous bacillus MCCC 1K03226chFull length gene.
It is further applied to line software Pro tParam (http://www.expasy.org/cgi-bin/protparam) Analyze its basic physicochemical property；Using online software PSORTb (http://www.psort.org/psortb/) carry out protein Subcellular Localization, it is known that the gene coded protein size (removal signal peptide) is 64.7kDa；Half-life period is 20h (external to feed In newborn zooblast), the coefficient of stability 40.17 belongs to degradable albumen；Protein Asia positioning analysis shows HlychAlbumen is extracellular Albumen, this biological function played with it are adapted, i.e., albumen is secreted bacterium, so as to cause host's erythrocyte hemolysis. Further DNAstar is utilized to analyze HlychProtein secondary structure, as a result such as Fig. 5.As shown in Figure 5, HlychProtein secondary structure packet Include 14 ɑ spirals, 22 β-pleated sheets and 50 corners.
3 recombinant expression carrier pET32a-hly of embodimentchStructure and prokaryotic expression
Using restriction enzyme BamH I and Sal I respectively to pMD18T-hlychIt is carried out with expression vector pET32a (+) Double digestion, gel electrophoresis recycle digestion products and according to 3:1 ratio mixing, 22 DEG C of connection 2h, large intestine is transformed by conjugate It in bacillus competence DH5 α, is coated on the LB tablets containing Amp, 37 DEG C of overnight incubations.Picking single bacterium colony shakes bacterium, logical using carrier PCR identifications are carried out with primer.Picking positive bacterium colony expands culture, preserves strain and extracts plasmid, recombinant plasmid is denoted as pET32a- hlych。
It is transferred in expression bacterial strain BL21 with the recombinant plasmid of extraction and is coated on the LB solid plates containing Amp, 37 DEG C were cultivated Night.Single bacterium colony on picking Amp tablets is shaken bacterium and is stayed overnight, and next day draws 1mL bacterium solutions and is inoculated in liquid LB cultures of the 100mL containing Amp Base, 220r/min shake bacterium, until OD600Value reaches 0.6, and IPTG induced expressions 4h is added.
4 Mermaid luminous bacillus hemolysin Hly of embodimentchThe identification method of albumen
Thalline were collected by centrifugation, and PBS is resuspended after PBS is washed 3 times, and Loading Buffer boiling lysis is added, and carries out SDS- PAGE, wherein a concentration of the 12% of separation gel, after electrophoresis, gel-colored decoloration observation, can by Fig. 6 as a result, result such as Fig. 6 Know, it is about 79kDa that destination protein, which meets expected albumen size, and the albumen of empty control plasmid (Empty vector) expression is about 20kDa。
Meanwhile using band His tag monoclonal antibodies as primary antibody, a concentration of 1:600-1000, sheep anti-mouse igg-HRP are two It is anti-, a concentration of 1:1000-2000 carries out the expression of Western blot verifying purpose albumen, and the results are shown in Figure 7, can by Fig. 7 Know occur expected trace band at 79kDa, Hly is obtained by Fig. 6 and Fig. 7chAlbumen is expressed.
Embodiment 5HlychThe sero-fast preparation in mouse source and titration of albumen
Induce HlychProtein expression, rear using ultrasound cracking thalline, centrifugation takes supernatant precipitation to carry out SDS- respectively PAGE determines HlychAlbumen is expressed for inclusion body；Using QIAGEN Ni-NTA columns to protein purification, purification result such as Fig. 8, by Fig. 8 is it is found that protein purification works well.
Freund's complete adjuvant, ratio 1 is added in purifying protein:1,6 week old BALB/c mouses, immunizing dose are immunized after emulsification Only for 100 μ g/；It is 1 that incomplete Freund's adjuvant ratio, which is added, in purifying protein after 3 weeks:1 is emulsified, and secondary immunity, then two are carried out Booster immunization after week, step are consistent with secondary immunity.Wherein secondary immunity and booster immunization dosage are 50 μ g/, and knot is immunized Eyeball of mouse takes blood after beam, serum is detached, by serum 1:After 200 dilutions, Western blot analyses are carried out, as a result such as Fig. 9.By For Fig. 9 it is found that occurring apparent trace band at about 79kDa, albumen is in the same size with being expected, and illustrates HlychAlbumen has one Fixed immunogenicity.
By the Mermaid luminous bacillus MCCC 1K03226 of culture to logarithmic phase, it is resuspended, is added using 1mL PBS washings After Loading Buffer boil sample, Western blot analyses are carried out.
With 10ng/ porin coated elisa plates, mouse immune serum is primary antibody, and HRP- goat-anti rabbit secondary antibodies are enzyme labelled antibody, ELISA measurement is carried out, antibody titer is measured.It is detected by ELISA, antibody titer 1:5000, illustrate HlychAlbumen have compared with High immunogenicity.
Embodiment 6HlychThe immune protective efficiency of albumen is evaluated
Immunization method is the same as embodiment 5：Freund's complete adjuvant, ratio 1 is added in purifying protein:1,6 week old are immunized after emulsification BALB/c mouse, immunizing dose are 100 μ g/；It is 1 that incomplete Freund's adjuvant ratio, which is added, in purifying protein after 3 weeks:1 carries out breast Change, carries out secondary immunity, then booster immunization after two weeks, step is consistent with secondary immunity, wherein secondary immunity and booster immunization agent Amount is 50 μ g/.Booster immunization after two weeks, carries out bacterium and attacks poison, Balb/c mouse Mermaid luminous bacillus is injected intraperitoneally MCCC 1K03226, dosage 5LD50(i.e. 6 × 107Cfu), the dead mouse quantity of statistics in latter week is infected.As shown in Figure 10, HlychAfter protein immunization mouse, immune protective efficiency is higher, can reach 65%.
In conclusion the Hly of Mermaid luminous bacillus of the present inventionchAlbumen has good immunogenicity and immunoprotection Power can be used for developing engineering subunit vaccine, lay a good foundation for the prevention and control of Mermaid luminous bacillus disease.
Each embodiment is described by the way of progressive in this specification, the highlights of each of the examples are with other The difference of embodiment, just to refer each other for identical similar portion between each embodiment.For device disclosed in embodiment For, since it is corresponded to the methods disclosed in the examples, so description is fairly simple, related place is said referring to method part It is bright.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, as defined herein General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one The widest range caused.
Sequence table
<110>Hebei Science ＆ Technology Normal College
<120>A kind of Mermaid luminous bacillus hemolysin Hlych albumen with immanoprotection action
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gctttagagc ttcttaattc tgaggatcta ataaagccag acccagaaga tcctagactt 180
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atcggtcaat atggcccaga ttgttttgat gcgacagcac aagaaccagc agatctccag 420
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gttcgattac cagaagataa ttataaaaaa attagacaag tcaaaggata tatttgttct 720
gacagttcag aacagcattc tcatagcagt tttacttctt ctgatttcta tgactctaat 780
gatgtaatat ggaagaaaaa atcaaaatct gataccggat ggcaatcata taaaccaagt 840
aatatgaagc cagccgcttt aactgcacta gaagcaagta aagatctatg ggctgctttt 900
ataagaacaa tggcaacaaa tattaatgaa cgggaaacta cagctagaaa agaggcacaa 960
caattaattg atacatggct atcttttgat gaggaagaaa tgttgtcatg gtatgacaac 1020
gaaaaaaatc gagatgatac atttgtaaca tctgataata aaaaaggaca atctcaaaaa 1080
acttgcttat caaacataag ctttaaaaat tcagatggtt caaagcctac acctactaac 1140
atagaagaat tagcatctaa tatagaaaag agtagaaata aatgtttatt taatattgaa 1200
cctgttcctg gtttcgctga tctatatgac ccttatatta aaatccctta taattggcag 1260
tggaaatcaa attcttggaa aacgcctggt caagattggg ctccatcaac cccaaaacct 1320
gataatggtg aagtcataaa tatattagcg tatgacaata atcagctttc agcagatact 1380
attgcaaata attcgaaaat tataacatca ggtaagaaag gtctagattt cataaaagta 1440
ccatcagaga ataatggtta ttatttcaga ctaaataatt accctaattt attctttagc 1500
tattcagcta gtgcagatgg aactgttaaa ttagtaaact cacctaaaca atcagagttt 1560
attttaatag gaaataaaaa cacatataac ataaagaaca catattggga tcagttcgta 1620
tggtataata aagctaaaaa atcagtacat ttaacgtctc atggtaacga aaaaaacaca 1680
gactcgtcat ggacaatatt aaataaagat attaataact ag 1722
<210> 2
<211> 1812
<212> DNA
<213> Artificial Sequence
<400> 2
atgaaaatta gaaaactata tagttgtata cttttaggtc ttagtagcct atccgctagt 60
gctatagccg aagttgataa ctattctaca cctgcagacg tagtgtcaat actaagcagt 120
gtaaaaaatc cagatcgtat tgtatatata aatatgaaac aggaggaact atcaacctac 180
gattctatat tagaagatat cataaataat gacaaacaat acatctttga tctttctttt 240
gatagtgatg aagaaaaagc taatcttcaa aagaaattta aggatttaat gggagtaaaa 300
tttgatagta attttatagt tgtaactggc tataaaaatc aactaatgta tactccgata 360
tcagatacta atgatcgtat gatatctatt ctagatcatg aagctaaatc taatgatata 420
tctaaattcc ctagagcttt agccttaagt tcaggtaaag ttgctagtga tcaatcaatt 480
tctttgcccc atgtttcgtt ttaccttaat gtcaataaag aaataactga tcaggaatgt 540
actttccgaa attctttaat ttgggataga ggaagtcgtt ctttctgtaa aaatggtaac 600
atttcactta tttatcaagt tatattagag cgttctttat catttggtac taatggcgtt 660
gcaactcctg atgcaaaaat agttcgaatt agcttagatg ataacactac tggtgccggt 720
atacatctaa atgatacact gacacataaa tcctattggg ctaattatca agtggtatct 780
ggttgggcta gagaatggtc agccagtgct attgctcaag attatttatt tgatatatcc 840
acttcaaatg aaaaagctca gatattaaaa acgttccctc gtgagaatat taattctaat 900
tataatataa atgaatcttc tggattcacc attggcgtta ctggtggtgc agaggtgagt 960
aaagatggac cgaaagctag tcttcaagct agtgctagtt atagtcaatc aaaatcatta 1020
tcttttaata cacaagatta tagagttgaa aagaattcaa cttctgcaca aaatgtttct 1080
ttccgttggg ctagagaaca atatcctaac tcagagtcat tgttagataa gtggacaaat 1140
cctgtatggt cagaagatta tcctgcaaat ctgaaaaaag ttcagcctct tagttatgcg 1200
agttttgttc ctaaacttga tgtcatatat aaagcttctc ctaatgaaac aggaaaaact 1260
caatttacta tagattcatc agttaatatt atgcctctat ataatagagc atggttctat 1320
ttttatggag ttggcgcaca ccagacctat tatggagtag atgaccaacc gcttcgtaga 1380
gttaacaaag caatatcatt tactgttgat tgggagcacc ctgtgtttac tggaggaacc 1440
ccagtaaata tacagttggc ttcttttaac aataaatgta tggatataca aaataaaggg 1500
atagtaatga ccgctgaatg tgatattaat agcaaatcgc agtcatttat ttatgatcaa 1560
tataatcgat acgttagtgc aactaatact aaactatgtt tggatggtga atctctatcg 1620
gaattacaac cttgtagttt gaagctaact caaaaatggt tatgggatgg agataaatta 1680
aaaaatagtt ttgaagataa atacttaact tttgataatg atactttctt attagaatct 1740
agcttatcta ataaacaaaa attaaattca tcatatacaa gagtttttga ttcatcgact 1800
ataaataagt aa 1812
<210> 3
<211> 1812
<212> DNA
<213> Artificial Sequence
<400> 3
atgaaaatta gaaaactata tagttgtata cttttaggtc ttagtagctt atctgccagc 60
gctatagctg atgttgataa ttattctacg ggcgcagacg ttatatcaat attaagcagt 120
gtgcaaaatc cagatcgtat tgtatatgta aatatgaagc aggaggaaat atcaacttac 180
aaccgtatat tagaagatat catatataat gataaacaat atatctttga tctttctttt 240
gataataatg aagaaaaaga gaagcttcaa aagaaattta aggatttaat gggagtaaaa 300
tttgatagta attttatagt tgtaacaggc tataaaaatc aactaatgta tactcctata 360
gcagatgcta atgatcgtat ggtatctgtt ctagatcatg aagccagatc taatgatatc 420
tctaaatacc caatggcttt agctttacgt tcaggtaacg ctactagtga tcaatcaatt 480
tctttgcccc atgtttcgtt ttatcttaat gttaataaag aaataactga tcaggaatgt 540
actttccgaa attctttaat ttgggataga ggaagtcgtt ctttctgtaa aaatggtaat 600
atttcactta tttatcaagt tatattagag cgttctttat cgtttggtac taatggcatt 660
gcaacgcctg atgcaaaaat agttcgaatt agcttagatg ataatacgac aggtgccggt 720
atacatctaa atgatacact gacgcaaaaa tattattggg ctaattatca ggtggtatct 780
ggttgggcta gagagtggtc agccagtgct attgctcaag attatttatt tgatatatct 840
acttcaaata aaaaagctca gatattaaaa acgttccctc gggagaatat taattctaat 900
tataatataa atgaatcttc tggattcacc attggtgtta ctggtggtgc tgaagttagt 960
aaagatggcc cgaaagcgag tcttcaggct aatgctagtt atagtcaatc taagtcgtta 1020
tcttttaata cccaagacta tagagttgaa aaaaattcaa cttctgccca aaatgtttct 1080
ttccgttggg cgagagagca gtatcctgac tcagagtcac tgttagataa gtggacaaac 1140
cctgtatggt cggaaggtta tcctgcgaat ctaaaaaaag ttcagccgct tagttatgcg 1200
agttttgttc ctaaacttga tgttatatac aaagcatctc ctaatgaaac aggaaaaact 1260
caatttacta tagattcatc agttaacatt atgcctctat ataatagatc gtggttctat 1320
ttttatggaa ttggtgcaca tcagacttat tatggtgtag atgatcagcc tcttcgtaga 1380
gttaataagg caatatcatt tactgttgat tgggagcacc ctgtatttac tggtggaact 1440
ccggtaaatt tacagttagc atcttttaat aataaatgta tcgatatcca aaacaacaat 1500
aaagtaatga cggctgaatg tgatattaat agtaaatcac agtcatttat ttatgaccaa 1560
tataatcgat atgttagcgc aactaatact aaattatgct tagatggcga atctttatca 1620
gaactgcaat cttgtagctt gaagctaacg caaaaatggt tatgggatgg agatagatta 1680
aaaaatagtt ttgaagataa atacttaact gttgatagtg atactttatc gttagaaact 1740
ggtttatcta ataaacaaaa attaaattca tcatatacaa gagtttttga cccatcaacc 1800
ataaatcagt aa 1812
<210> 4
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 4
taactcctat gggacatgaa tgg 23
<210> 5
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 5
tatagatcag cgaaaccagg aac 23
<210> 6
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 6
agcctcttag ttatgcgagt t 21
<210> 7
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 7
cggtcattac tatcccttta tt 22
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 8
atttactgtt gattgggagc 20
<210> 9
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 9
gatttatggt tgatgggt 18
<210> 10
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 10
gcggatccga cgttatatca atattaagca g 31
<210> 11
<211> 32
<212> DNA
<213> Artificial Sequence
<400> 11
gcgtcgactt actgatttat ggttgatggg tc 32

Claims (10)

1. a kind of Mermaid luminous bacillus hemolysin Hly with immanoprotection actionchAlbumen, in exploitation Mermaid luminous bacillus It is applied in genetic engineering subunit vaccine.
2. a kind of Mermaid luminous bacillus hemolysin Hly with immanoprotection action according to claim 1chAlbumen Preparation method, which is characterized in that be as follows：
1) recombinant vector pMD18T-hlychStructure
The genomic DNA of Mermaid luminous bacillus is extracted, the culture presevation number of the Mermaid luminous bacillus is MCCC1K03226, using genomic DNA as template, with Hlych-F、Hlych- R is that primer amplification removes signal coding sequence hlychGene, the hlychGene order is SEQ ID NO：3；Hly is expanded using touchdown PCRchFull length gene, the primer Hlych-F、HlychThe sequence of-R is as follows：
Hlych-F：5'-GCGGATCCGACGTTATATCAATATTAAGCAG-3'；With I restriction enzyme sites of BamH；SEQ ID NO： 10；
Hlych-R：5'-GCGTCGACTTACTGATTTATGGTTGATGGGTC-3'；With Sal I restriction enzyme sites；SEQ ID NO：11；
By the hly of acquisitionchGene is connect with carrier T, obtains recombinant vector pMD18T-hlych, and to the recombinant vector pMD18T-hlychIt is sequenced, carries out proteins subcellular location；
2) recombinant expression carrier pET32a-hlychStructure
Using restriction enzyme BamH I and Sal I respectively to the recombinant vector pMD18T-hlychAnd expression vector PET32a (+) carries out double digestion, and gel electrophoresis is recycled digestion products, mixed in proportion, and 22-25 DEG C of connection 2-3h converts large intestine In bacillus competent cell, in access LB tablets one, 37 DEG C of overnight incubations；Picking single bacterium colony shakes bacterium, uses carrier universal primer Carry out PCR identifications；Positive bacterium colony is expanded and is cultivated, strain is preserved and extracts plasmid pET32a-hlych；
3)hlychThe prokaryotic expression of gene
By the plasmid pET32a-hlychIn conversion expression bacterial strain BL21, it is applied in LB tablets two, 37 DEG C of overnight incubations；It chooses It taking the single bacterium colony of the LB tablets two to shake bacterium to stay overnight, bacterium solution is inoculated into LB liquid medium by next day, and 220r/min shakes bacterium, until OD600IPTG induced expressions 4-6h is added up to being 0.6-0.8 in value.
3. the Mermaid luminous bacillus hemolysin Hly according to claim 2 with immanoprotection actionchThe preparation of albumen Method, which is characterized in that recombinant vector pMD18T-hly described in step 2)chIt is returned with expression vector pET32a (+) digestion The ratio for receiving product is 3:1.
4. a kind of Mermaid luminous bacillus hemolysin Hly with immanoprotection action according to claim 2chAlbumen Preparation method, which is characterized in that competent cell described in step 2) is DH5 α.
5. a kind of Mermaid luminous bacillus hemolysin Hly with immanoprotection action according to claim 2chAlbumen Preparation method, which is characterized in that LB tablets described in step 2) one is the solid containing ampicillin with the LB tablets two LB tablets, a concentration of 45-55 μ g/L of the ampicillin.
6. a kind of Mermaid luminous bacillus hemolysin Hly with immanoprotection action according to claim 2chAlbumen Preparation method, which is characterized in that liquid LB described in step 3) is the liquid LB containing ampicillin, the ampicillin A concentration of 45-55 μ g/L.
7. a kind of Mermaid luminous bacillus hemolysin Hly with immanoprotection action according to claim 2chAlbumen Preparation method, which is characterized in that a concentration of 1mM of IPTG described in step 3).
8. a kind of Mermaid luminous bacillus hemolysin Hly with immanoprotection actionchThe identification method of albumen, feature exist In, including SDS-PAGE analyses and Western blot verifications；
Bacteria suspension in claim 1 step 3) is centrifuged and is collected thalline, PBS is resuspended after washing 3 times, and Loading is added Buffer boiling lysis carries out SDS-PAGE electrophoresis, after electrophoresis, gel-colored decoloration observation result；It is carried out at the same time The expression of Western blot verifying purpose albumen.
9. a kind of Mermaid luminous bacillus hemolysin Hly with immanoprotection action according to claim 8chAlbumen Identification method, which is characterized in that resolving gel concentration is 12% in the SDS-PAGE electrophoresis.
10. a kind of Mermaid luminous bacillus hemolysin Hly with immanoprotection action according to claim 8chAlbumen Identification method, which is characterized in that in the Western blot methods, primary antibody is band His tag monoclonal antibodies, secondary antibody is Sheep anti-mouse igg-HRP.
CN201810132388.9A 2018-02-09 2018-02-09 Mermaid photorhabditis hemolysin Hly with immune protection effectchProtein Active CN108330142B (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109395071A (en) * 2018-11-16 2019-03-01 河北科技师范学院 A kind of Mermaid luminous bacillus extracellular protein EF-Tu
CN109880766A (en) * 2019-03-18 2019-06-14 宁波大学 A kind of silvery pomfret Mermaid luminous bacillus bacterial strain and inactivated vaccine
CN111304131A (en) * 2020-03-17 2020-06-19 中国水产科学研究院黄海水产研究所 Strong-pathogenicity mermaid photobacterium mermaid subspecies strain and application thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1786022A (en) * 2005-10-11 2006-06-14 中山大学 Mermaid luminous bacillus outer membrane protein V and coding sequence, its preparation method and application
CN1800209A (en) * 2005-10-11 2006-07-12 中山大学 Mermaid photobacterium envelop protein W and coding sequence , its preparation method and uses
CN101461940A (en) * 2009-01-09 2009-06-24 中国水产科学研究所南海水产研究所 Photobacterium damsela vaccine as well as preparation method and use thereof
JP5567244B2 (en) * 2006-06-06 2014-08-06 共立製薬株式会社 Inactivated vaccine with fish Streptococcus disgalactie as antigen
US20150010592A1 (en) * 2010-05-06 2015-01-08 Glycovaxyn Ag Capsular gram-positive bacteria bioconjugate vaccines

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1786022A (en) * 2005-10-11 2006-06-14 中山大学 Mermaid luminous bacillus outer membrane protein V and coding sequence, its preparation method and application
CN1800209A (en) * 2005-10-11 2006-07-12 中山大学 Mermaid photobacterium envelop protein W and coding sequence , its preparation method and uses
JP5567244B2 (en) * 2006-06-06 2014-08-06 共立製薬株式会社 Inactivated vaccine with fish Streptococcus disgalactie as antigen
CN101461940A (en) * 2009-01-09 2009-06-24 中国水产科学研究所南海水产研究所 Photobacterium damsela vaccine as well as preparation method and use thereof
US20150010592A1 (en) * 2010-05-06 2015-01-08 Glycovaxyn Ag Capsular gram-positive bacteria bioconjugate vaccines

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AMABLE J.RIVAS ET AL: "Synergistic and Additive Effects of Chromosomal and Plasmid-Encoded Hemolysins Contribute to Hemolysis and Virulence in Photobacterium damselae subsp. damselae", 《INFECTION AND IMMUNITY》 *
窦骏 等: "《疫苗工程学》", 31 August 2014, 东南大学出版社 *
赵秀敏: "嗜水气单胞菌溶血素A片段基因的克隆、表达及其产物的免疫学特性", 《中国优秀硕士学位论文全文数据库 农业科技辑》 *
马文丽 等: "《分子生物学实验手册》", 30 June 2011, 人民军医出版社 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109395071A (en) * 2018-11-16 2019-03-01 河北科技师范学院 A kind of Mermaid luminous bacillus extracellular protein EF-Tu
CN109395071B (en) * 2018-11-16 2021-08-06 河北科技师范学院 Mecanus amabilis extracellular protein EF-Tu
CN109880766A (en) * 2019-03-18 2019-06-14 宁波大学 A kind of silvery pomfret Mermaid luminous bacillus bacterial strain and inactivated vaccine
CN111304131A (en) * 2020-03-17 2020-06-19 中国水产科学研究院黄海水产研究所 Strong-pathogenicity mermaid photobacterium mermaid subspecies strain and application thereof

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