CN102978219A - Vibrio cross-protective antigen, and preparation and application thereof - Google Patents
Vibrio cross-protective antigen, and preparation and application thereof Download PDFInfo
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Abstract
The invention relates to a vibrio cross-protective antigen, and a preparation and an application thereof. The antigen is vibrio recombinant maltoporin LamB. With the preparation method provided by the invention, recombinant maltoporin LamB with natural immunocompetence can be expressed in a large amount; purification is easy; and cost is low. As an outer membrane major protein, the LamB protein widely exists in various vibrios. The sequence has good conservativeness. As an antigen, the protein has cross-protectiveness upon various vibrios in a zebrafish immune model. A rabbit antiserum of the protein has cross-reactivity and passive immune protection effect upon various vibrios. Therefore, the recombinant maltoporin LamB provided by the invention can be applied as a vibrio cross-protective antigen in aquaculture.
Description
Technical field
The present invention relates to genetically engineered and field of immunology, specifically, relate to a kind of vibrios intersecting protective antigen and its preparation method and application.
Background technology
Vibrio (Vibrio) is distributed widely in nature, is many among the You Yishui, and kind more than 100 is arranged.Main Pathogenic Bacteria can cause cholera or food poisoning.
The outer membrane protein of vibrios plays very important effect at its pathogenic course, is a kind of good immunogen; Porin is as one of main outer membrane protein, and the popular research object that is the vibrios intersecting protective vaccine also is the efficient immunogenic popular research object of vibrios.Some porins such as OmpK, OmpU and OmpV, its gene order has good conservative property in vibrios, during as antigen, have good immunogenicity and immune protective, and OmpK is proved to be especially and has good intersecting protective.
Maltoporin (maltoporin) is a porin relevant with the transportation of maltose, maltodextrin.Acceptor property called after LamB according to its phage.The physiological function of LamB protein and protein structure research and comparison are ripe, but its immunological properties research is less, does not see LamB albumen as the relevant report of antigen.Estimate the conservative property of vibrios LamB gene order, immunogenicity and cross immunity provide protection will help to inquire into exploitation vibrios maltoporin LamB as the using value of intersecting protective antigen.
Summary of the invention
The purpose of this invention is to provide a kind of vibrios intersecting protective antigen and its preparation method and application.
In order to realize purpose of the present invention; the present invention prepares the maltoporin encoding gene LamB of vibrios through gene clone, prokaryotic expression and product; the intersecting protective antigen that the maltoporin LamB that obtains recombinating, this recombinant protein can be used as vibrios is applied in the aquaculture.
The purpose of this invention is to provide a kind of maltoporin encoding gene LamB, this gene has the nucleotide sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8.
The present invention is according to existing LamB sequence, and the design degenerated primer has obtained vibrio alginolyticus ATCC33787(Vibrio alginolyticus), Vibrio parahaemolyticus ATCC17802(Vibrio parahaemolyticus), Vibrio parahaemolyticus VPL4-90(Vibrio parahaemolyticusVPL4-90), Vibrio mimicus ATCC33653(Vibrio mimicus), non-zero 1 vibrio cholerae (Vibrio cholerae Vb0), Vibrio flurialis ATCC33810(Vibrio fluvialis), Vibrio furnissii ATCC33813(Vibrio furnissii) and Vibrio harveyi NBRC15634(Vibrio harveyi) gene LamB.
The degenerated primer sequence is as follows:
LamB-F:5'-ATGAAAAAAGTAAGTSNYATTGCAG-3'
LamB-R:5'-TTACCACCAAGCTTCNRCTTG-3'
Wherein, S=C/G; N=A/C/G/T; Y=C/T; R=A/G;
Wherein, the nucleotide sequence of the gene LamB of vibrio alginolyticus ATCC33787 is shown in SEQID NO.1, the nucleotide sequence of the gene LamB of Vibrio parahaemolyticus ATCC17802 is shown in SEQ ID NO.2, the nucleotide sequence of the gene LamB of Vibrio parahaemolyticus VPL4-90 is shown in SEQ ID NO.3, the nucleotide sequence of the gene LamB of Vibrio mimicus ATCC33653 is shown in SEQ ID NO.4, the nucleotide sequence of the gene LamB of non-zero 1 vibrio cholerae is shown in SEQ ID NO.5, the nucleotide sequence of the gene LamB of Vibrio flurialis ATCC33810 is shown in SEQ ID NO.6, the nucleotide sequence of the gene LamB of Vibrio furnissii ATCC33813 is shown in SEQ ID NO.7, and the nucleotide sequence of the gene LamB of Vibrio harveyi NBRC15634 is shown in SEQ ID NO.8.
Vibrio alginolyticus ATCC33787, Vibrio parahaemolyticus ATCC17802, Vibrio mimicus ATCC33653, Vibrio flurialis ATCC33810, Vibrio furnissii ATCC33813 and Vibrio harveyi NBRC15634 six strain vibrios are reference culture, are preserved in US mode culture collection warehousing ATCC and the Japanese technological assessment Biological resources center NBRC of institute.Vibrio parahaemolyticus VPL4-90 and non-zero 1 vibrio cholerae are bought in Huankai Microbes Tech Co., Ltd., Guangdong.
The purpose of this invention is to provide a kind of maltoporin LamB, this albumen is encoded by above-mentioned maltoporin encoding gene.
The purpose of this invention is to provide a kind of LamB albumen that derives from the maltoporin encoding gene LamB coding of vibrio alginolyticus ATCC33787, this maltoporin has the aminoacid sequence shown in SEQ ID NO.9.
The purpose of this invention is to provide the preparation method of a kind of maltoporin LamB that recombinates, the method comprises the steps:
(1) clone of maltoporin encoding gene LamB;
(2) structure of coli expression carrier pET32-LamB;
(3) structure of intestinal bacteria recombinant bacterium BL21-pET32-LamB;
(4) preparation of restructuring maltoporin LamB.
Concrete, the preparation method of a kind of maltoporin LamB that recombinates provided by the invention, the method comprises the steps:
(1) clone of maltoporin encoding gene LamB: the design degenerated primer, take the genomic dna of vibrios as template, carry out PCR, obtain maltoporin encoding gene LamB; Then be connected among the pMD19-T, make up escherichia coli cloning carrier pMD19-T-LamB; This carrier is converted into DH5 α competent cell, makes up intestinal bacteria recombinant bacterium DH5 α-pMD19-T-LamB, order-checking; Wherein the degenerated primer sequence is as follows:
LamB-F:5'-ATGAAAAAAGTAAGTSNYATTGCAG-3'
LamB-R:5'-TTACCACCAAGCTTCNRCTTG-3'
Wherein, S=C/G; N=A/C/G/T; Y=C/T; R=A/G;
(2) structure of coli expression carrier pET32-LamB: primer is expressed in design, take cloning vector pMD19-T-LamB as template, carry out PCR, the LamB gene and the expression vector pET32a (+) that obtain are carried out double digestion with BamH I and Xho I, connect, make up coli expression carrier pET32-LamB; It is as follows wherein to express primer sequence:
LamBeF:5'-CGCGGATCCGCAGTGGATTTTAAC-3'
LamBeR:5'-CCGCTCGAGATTCGGCTTTTACCAC-3'；
(3) structure of intestinal bacteria recombinant bacterium BL21-pET32-LamB: the coli expression carrier pET32-LamB in (2) is transformed the BL21 competent cell, obtain intestinal bacteria recombinant bacterium BL21-pET32-LamB;
(4) preparation of restructuring maltoporin LamB: contain the penbritin of 100 μ g/ml at liquid LB() access intestinal bacteria recombinant bacterium BL21-pET32-LamB bacterium liquid in (3) of overnight incubation in the substratum by 1% ratio, add 1mM isopropylthio-β-D-galactoside (IPTG) at 37 ℃ after being cultured to OD600=0.6, carry out abduction delivering 6h in 37 ℃ of temperature; Collect thalline, purifying inclusion body, inclusion body purification liquid are directly crossed nickel NTA sepharose FF post, the affinitive layer purification recombinase, and imidazoles gradient competitive elution obtains the restructuring maltoporin LamB of purifying.
The purpose of this invention is to provide a kind of restructuring maltoporin LamB by the aforesaid method preparation, the intersecting protective antigen that described restructuring maltoporin LamB can be used as vibrios is applied in the aquaculture.
The degenerated primer that the purpose of this invention is to provide preparation restructuring maltoporin LamB:
LamB-F:5'-ATGAAAAAAGTAAGTSNYATTGCAG-3'
LamB-R:5'-TTACCACCAAGCTTCNRCTTG-3'；
Wherein, S=C/G; N=A/C/G/T; Y=C/T; R=A/G.
The expression primer that the purpose of this invention is to provide preparation restructuring maltoporin LamB:
LamBeF:5'-CGCGGATCCGCAGTGGATTTTAAC-3'
LamBeR:5'-CCGCTCGAGATTCGGCTTTTACCAC-3'。
The escherichia coli cloning carrier pMD19-T-LamB that the purpose of this invention is to provide preparation restructuring maltoporin LamB.
The intestinal bacteria recombinant bacterium DH5 α-pMD19-T-LamB that the purpose of this invention is to provide preparation restructuring maltoporin LamB.
The coli expression carrier pET32-LamB that the purpose of this invention is to provide preparation restructuring maltoporin LamB.
The intestinal bacteria recombinant bacterium BL21-pET32-LamB that the purpose of this invention is to provide preparation restructuring maltoporin LamB.
Particularly, the purpose of this invention is to provide a kind of escherichia coli cloning carrier that contains aforementioned maltoporin encoding gene LamB.
Preferably, above-mentioned cloning vector is a kind of escherichia coli cloning carrier pMD19-T-LamB, and this carrier contains maltoporin encoding gene LamB.That is, with the gene LamB of vibrio alginolyticus ATCC33787, Vibrio parahaemolyticus ATCC17802, Vibrio parahaemolyticus VPL4-90, Vibrio mimicus ATCC33653, non-zero 1 vibrio cholerae, Vibrio flurialis ATCC33810, Vibrio furnissii ATCC33813 and Vibrio harveyi NBRC15634 for the preparation of escherichia coli cloning carrier pMD19-T-LamB.
The purpose of this invention is to provide a kind of host cell that contains above-mentioned cloning vector.
Preferably, the above-mentioned host cell that contains cloning vector is intestinal bacteria recombinant bacterium DH5 α-pMD19-T-LamB, and this recombinant bacterium contains escherichia coli cloning carrier pMD19-T-LamB.That is, will contain the cloning vector pMD19-T-LamB of encoding gene LamB of vibrio alginolyticus ATCC33787, Vibrio parahaemolyticus ATCC17802, Vibrio parahaemolyticus VPL4-90, Vibrio mimicus ATCC33653, non-zero 1 vibrio cholerae, Vibrio flurialis ATCC33810, Vibrio furnissii ATCC33813 and Vibrio harveyi NBRC15634 for the preparation of intestinal bacteria recombinant bacterium DH5 α-pMD19-T-LamB.
The purpose of this invention is to provide a kind of coli expression carrier that contains aforementioned maltoporin encoding gene LamB.That is, take aforementioned escherichia coli cloning carrier pMD19-T-LamB as template, the design primer carries out PCR, makes up coli expression carrier pET32-LamB.
The purpose of this invention is to provide a kind of host cell that contains above-mentioned expression vector, this host cell is intestinal bacteria recombinant bacterium BL21-pET32-LamB, can Expression product restructuring maltoporin.
Preferably, the purpose of this invention is to provide a kind of coli expression carrier that contains the maltoporin encoding gene LamB of vibrio alginolyticus ATCC33787.This coli expression carrier pET32-LamB contains the maltose protein coding gene LamB of vibrio alginolyticus ATCC33787.That is, take the escherichia coli cloning carrier pMD19-T-LamB of the gene LamB that contains vibrio alginolyticus ATCC33787 as template, make up coli expression carrier pET32-LamB.
Preferably, the host cell that the purpose of this invention is to provide the coli expression carrier of a kind of gene LamB that contains vibrio alginolyticus ATCC33787, be intestinal bacteria recombinant bacterium BL21-pET32-LamB, to contain the expression vector pET32-LamB of encoding gene LamB of vibrio alginolyticus ATCC33787 for the preparation of intestinal bacteria recombinant bacterium BL21-pET32-LamB, this recombinant bacterium can Expression product restructuring maltoporin LamB.
The purpose of this invention is to provide the application of described restructuring maltoporin LamB in preparation vibrios intersecting protective vaccine.
The purpose of this invention is to provide a kind of vibrios intersecting protective vaccine, this vaccine contains described restructuring maltoporin LamB.After inoculating described vibrios intersecting protective vaccine, body can obtain the protectiveness to Vibrio.
The purpose of this invention is to provide the application of described restructuring maltoporin LamB in preparation treatment vibriosis medicine.
The purpose of this invention is to provide the medicine for the treatment of vibriosis, this medicine contains described restructuring maltoporin LamB.
The purpose of this invention is to provide described restructuring maltoporin LamB uses in Dispersal risk.
The purpose of this invention is to provide described restructuring maltoporin LamB is the antibody of immunogen preparation.
Antibody can be obtained by the antiserum(antisera) purifying that restructuring maltoporin LamB immune rabbit obtains.
The purpose of this invention is to provide a kind of rabbit anti-serum by described restructuring maltoporin LamB preparation.This rabbit anti-serum has cross reactivity to Vibrio.
The preparation method who the purpose of this invention is to provide a kind of described rabbit anti-serum: adjust restructuring maltoporin LamB to final concentration of protein be 2mg/mL, the recombinant protein solution of handling well is mixed with isopyknic freund's adjuvant, and whirlpool shakes the agent of fully emulsified one-tenth " water-in-oil " emulsus.Authentication method is that emulsifying agent is splashed in the cold water, does not disperse if keep complete, becomes to drip shape and bubbles through the water column, i.e. emulsification is complete, is qualified water-in-oil agent.With the immunity of the recombinant protein LamB after emulsification new zealand white rabbit.During first immunisation, recombinant protein LamB and isopyknic Freund's complete adjuvant emulsification, the subcutaneous multi-point injection in back, every two all booster immunizations once, and during booster immunization, recombinant protein LamB and Freund's incomplete adjuvant emulsification.For the second time behind the booster immunization the 8th day by ear edge arterial blood extracting 1mL, measure serum titer.After reaching satisfied tiring, the carotid artery bloodletting.Centrifugation serum, tubule packing, freezing preservation.
The purpose of this invention is to provide above-mentioned antibody for the preparation of the application in the diagnostic reagent that detects vibriosis.
The purpose of this invention is to provide the diagnostic reagent for detection of vibriosis that contains above-mentioned antibody.
The purpose of this invention is to provide above-mentioned antibody for the preparation of the application in the diagnostic kit that detects vibriosis.
The purpose of this invention is to provide the diagnostic kit for detection of vibriosis that contains above-mentioned antibody.This diagnostic kit contains aforementioned diagnostic reagent.
Above-mentioned diagnostic reagent and diagnostic kit are to utilize its antibody that contains, and can detect vibrios through the ELISA method.
Another object of the present invention provides medicine, antibody, diagnostic reagent and the diagnostic kit application in estimating vibrios LamB gene order conservative property, restructuring maltoporin LamB immunogenicity and cross immunity provide protection of described vaccine, treatment vibriosis.
Advantage of the present invention is as follows:
1) the present invention utilizes genetic engineering technique to obtain a kind of restructuring maltoporin LamB, and this recombinant protein can prepare medicine, antibody, diagnostic reagent and the diagnostic kit of vibrios intersecting protective vaccine, treatment vibriosis.
2) rabbit anti-serum of recombinant protein LamB preparation has cross reactivity and passive immunization provide protection to Vibrio; recombinant protein LamB itself is as antigen; take zebra fish in immune model; therefore Vibrio is had intersecting protective, and the recombinant protein LamB of the present invention intersecting protective antigen that can be used as vibrios is applied in the actual cultivation.
3) intestinal bacteria recombinant bacterial strain BL21-pET32-LamB of the present invention can a large amount of expression has the restructuring LamB albumen of innate immune activity, is easy to purifying, can be applicable to produce the vibrios intersecting protective vaccine, and cost is low.
4) medicine, antibody, diagnostic reagent and the diagnostic kit of vibrios intersecting protective vaccine provided by the invention, treatment vibriosis are to utilize first restructuring maltoporin LamB preparation; to help to inquire into the using value that exploitation vibrios maltoporin LamB prepares medicine, antibody, diagnostic reagent and the diagnostic kit of vibrios intersecting protective vaccine, treatment vibriosis, significant for conservative property, immunogenicity and the cross immunity provide protection of estimating vibrios LamB gene order.
Description of drawings
Fig. 1 is vibrios LamB gene nucleotide series phylogenetic tree;
Wherein, V.fluvialis ATCC33810 (JF747209) Vibrio flurialis ATCC33810, V.furnissii ATCC33813 (JF747208) is Vibrio furnissii ATCC33813, V.furnissii NCTC11218 (CP002378.1) is Vibrio furnissii NCTC11218, V.cholerae MJ-1236 (229605062) is vibrio cholerae MJ-1236, V.cholerae (CP001486.1) is vibrio cholerae, V.cholerae Vb0(JF747210) is non-zero 1 vibrio cholerae, V.mimicus ATCC33653 (JF747206) is Vibrio mimicus ATCC33653, V.alginolyticus ATCC33787 (JF747203) is vibrio alginolyticus ATCC33787, V.parahaemolyticus ATCC17802 (JF747207) is Vibrio parahaemolyticus ATCC17802, V.harveyi NBRC15634 (JF747211) is Vibrio harveyi NBRC15634, V.harveyi ATCC BAA-1116 (CP000790.1) is Vibrio harveyi ATCCBAA-1116, V.alginolyticus (EU625279.1) is vibrio alginolyticus, Vibriosp.Ex25 (CP001806.1) is the Ex25 vibrios, V.parahaemolyticus VPL4-90(JF904926.1) is Vibrio parahaemolyticus VPL4-90, Cluster A is gene cluster A, ClusterB is gene cluster B, Cluster C is gene cluster C, is the number of logining of the NCBI sequence of each bacterial strain in the bracket;
Fig. 2 is the SDS-PAGE electrophoresis result of the restructuring maltoporin LamB of purifying in the embodiment of the invention 4;
Wherein, M is albumen Marker, and 1 is the restructuring maltoporin LamB of purifying, and its molecular weight is 59.4kDa;
Fig. 3 is the Western blotting analytical results of rabbit anti-serum of the restructuring maltoporin LamB of the embodiment of the invention 5;
Wherein, 1 is extra large vibrios (V.pelagius); 2 is Vibrio natriegen (V.natriegens); 3 is Vibrio furnissii (V.furnissii); 4 is Vibrio flurialis (V.fluvialis); 5 is Vibrio mimicus (V.mimicus); 6 is Vibrio vulnificus (V.vulnificus); 7 is vibrio alginolyticus (V.alginolyticus); 8 is vibrio cholerae (V.cholera); 9 is Vibrio parahaemolyticus (V.parahaemolyticus); 10 is Vibrio parahaemolyticus VPL4-90(V.parahaemolyticus4-90); 11 is vibrio proteolyticus (V.proteolyticus); 12 is bacillus coli DH 5 alpha (E.coliDH5 α); 13 is streptococcus aureus (S.aureus); 14 is subtilis (B.subtilis); 15 is aeromonas hydrophila (A.hydrophilic); 16 is Vibrio anguillarum (V.anguillarum); 17 is Vibrio splindidus (V.splendidus); 18 is Vibrio campbellii (V.campbellii); 19 is V.metsehnikvii (V.metschnikovii); 20 is Vibrio harveyi (V.harveyi); 21 is mermaid vibrios (V.damsela); 22 is Fei Shi vibrios (V.fischeri).
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.
Reagent or experiment material used among the present invention are conventional molecular biology experiment with reagent or experiment material, are the commercially available prod.
The clone of embodiment 1 maltoporin gene LamB
Maltoporin precursor protein (Maltoporin according to Vibrio parahaemolyticus (Vibrio parahaemolyticus) RIMD2210633, being called for short LamB) (accession number is gene order: NP_801154), comprehensive other vibrios LamB gene orders, the design degenerated primer, synthetic primer.Take the genomic dnas of eight strain vibrios as template, carry out PCR respectively.The degenerated primer sequence is as follows:
LamB-F:5'-ATGAAAAAAGTAAGTSNYATTGCAG-3'
LamB-R:5'-TTACCACCAAGCTTCNRCTTG-3'
Wherein, S=C/G; N=A/C/G/T; Y=C/T; R=A/G;
Reaction system (25 μ L): 1 μ LDNA template, 1 μ L forward primer, 1 μ L reverse primer, 9.5 μ L distilled waters, 12.5 μ L2 * Taq PCR Master mix.
Reaction conditions: 95 ℃ of denaturation 5min, 95 ℃ of 30s then, 58 ℃ of 45s, 72 ℃ of 1min, 30 circulations.The whole 10min that extends after reaction finishes.
The PCR product is obtained maltoporin gene LamB by the test kit recovery; Then be connected among the pMD19-T, make up the sub-pMD19-T-LamB of recombinant clone; Be converted into DH5 α competent cell, be coated with at the LB agar plate that contains Amp and isopropylthio-β-D-galactoside (IPTG), X-gal that presets, by blue hickie screening positive clone, order-checking after the PCR checking.The open reading frame that sequencing result is removed after the carrier sequence is:
The nucleotide sequence of the gene LamB of vibrio alginolyticus ATCC33787 is shown in SEQ IDNO.1; The nucleotide sequence of the gene LamB of Vibrio parahaemolyticus ATCC17802 is shown in SEQ ID NO.2; The nucleotide sequence of the gene LamB of Vibrio parahaemolyticus VPL4-90 is shown in SEQ ID NO.3; The nucleotide sequence of the gene LamB of Vibrio mimicus ATCC33653 is shown in SEQ ID NO.4; The nucleotide sequence of the gene LamB of non-zero 1 vibrio cholerae is shown in SEQ ID NO.5; The nucleotide sequence of the gene LamB of Vibrio flurialis ATCC33810 is shown in SEQ ID NO.6; The nucleotide sequence of the gene LamB of Vibrio furnissii ATCC33813 is shown in SEQ ID NO.7; The nucleotide sequence of the gene LamB of Vibrio harveyi NBRC15634 is shown in SEQ IDNO.8.
Vibrios LamB gene nucleotide series phylogenetic tree as shown in Figure 1.
The structure of embodiment 2 coli expression carrier pET32-LamB
By the LamB gene clone sequence of embodiment 1 resulting vibrio alginolyticus, the expression primer of signal peptide is removed in design, synthetic this primer, and primer sequence is as follows:
LamBeF:5'-CGCGGATCCGCAGTGGATTTTAAC-3'
LamBeR:5'-CCGCTCGAGATTCGGCTTTTACCAC-3'
Take by embodiment 1 resulting LamB recombinant clone plasmid pMD19-T-LamB as template, carry out PCR, reaction conditions is as follows:
Reaction system (25 μ L): 1 μ LDNA template, 1 μ L forward primer, 1 μ L reverse primer, 9.5 μ L distilled waters, 12.5 μ L2 * Taq PCR Master mix.
Reaction conditions: 95 ℃ of denaturation 5min, 95 ℃ of 30s then, 58 ℃ of 45s, 72 ℃ of 1min, 30 circulations.The whole 10min that extends after reaction finishes.
The PCR product is reclaimed, and it is as follows to use resulting LamB gene and expression vector pET32a (+) to carry out the double digestion condition with BamH I and Xho I respectively:
Reaction system (20 μ L): distilled water 10 μ L, 10 * K Buffer2 μ L, gene LamB (perhaps pET32a (+) plasmid DNA) 6 μ L, BamH1 μ L, Xho I 1 μ L cut 2 hours at 37 ℃ of lower enzymes.Reclaim respectively the purpose fragment that size is about 1.2kb and 5.9kb behind the electrophoresis.The two is respectively LamB gene and expression vector pET32a (+).
Then with the T4 ligase enzyme the two is connected, reaction conditions is as follows:
Reaction system (10 μ L): 1.2kb fragment 5 μ L, 5.9kb fragment 3 μ L, 10 * T4 ligase enzyme Buffer1 μ L, T4 ligase enzyme 1 μ L connects more than 8 hours at 16 ℃ of lower enzymes.Namely can obtain coli expression carrier pET32-LamB.
The structure of embodiment 3 intestinal bacteria recombinant bacterium BL21-pET32-LamB
Get the coli expression carrier pET32-LamB5 μ L that is obtained by embodiment 2 and join in the 50 μ L BL21 competent cells, place 30min on ice; Then put into 42 ℃ of heat shocks 90 seconds; Fast the EP pipe is transferred to 15min in the ice bath; Then every pipe adds 350 μ L LB substratum, and pipe is transferred to 37 ℃ of shaking tables, and slowly jolting 60min makes bacteria resuscitation; The bacterium liquid of getting after 50 μ L recover is coated on the solid LB flat board that contains Amp and X-Gal and IPTG; Be inverted plate, in 37 ℃ of constant temperature culture 24h; The single bacterium colony of last picking white is used for PCR and detects, and obtains intestinal bacteria recombinant bacterium BL21-pET32-LamB.
The preparation of embodiment 4 restructuring maltoporin LamB
Contain the penbritin of 100 μ g/ml at liquid LB() in the substratum in the intestinal bacteria recombinant bacterium BL21-pET32-LamB bacterium liquid that is obtained by embodiment 3 of 1% ratio access overnight incubation, add 1mM isopropylthio-β-D-galactoside (IPTG) at 37 ℃ after being cultured to OD600=0.6, carry out abduction delivering 6h in 37 ℃ of temperature.Collect thalline, the purifying inclusion body, because recombinant protein contains 6 * HIS label, inclusion body purification liquid is directly crossed nickel NTA sepharose FF post, the affinitive layer purification recombinase, imidazoles gradient competitive elution, purge process is with reference to HIS purifying Kit specification sheets.Agarase behind the purifying utilizes the BCA method to measure protein concentration, by detect purity and the molecular weight of restructuring LamB albumen with 12%SDS-PAGE, the results are shown in Figure 2.The restructuring maltoporin LamB of purifying is stored in-20 ℃ with for subsequent use, and this restructuring maltoporin LamB is vibrios intersecting protective antigen.
The cross-immunoreactivity analysis of embodiment 5 restructuring maltoporin LamB
Animal rearing: after new zealand white rabbit is bought, raise a week at biology department's Animal House, make it adapt to laboratory condition.Every day is with laboratory animal rabbit feed feeding 2 times, sooner or later each once, each feeding amount is eaten up with about 15min and is advisable.
Immunogenic preparation: the restructuring LamB albumen of the purifying that is obtained by embodiment 4, adopt the Bradford method to measure its protein concentration, and to be adjusted to final concentration of protein be 2mg/mL, namely can be used as immunizing antigen and use.Antigen protein solution after the processing is for subsequent use in 4 ℃ of Refrigerator stores.The recombinant protein solution of handling well mixes with isopyknic freund's adjuvant, and whirlpool shakes the agent of fully emulsified one-tenth " water-in-oil " emulsus.Authentication method is that emulsifying agent is splashed in the cold water, does not disperse if keep complete, becomes to drip shape and bubbles through the water column, i.e. emulsification is complete, is qualified water-in-oil agent.
The preparation of rabbit anti-serum: with the immunity of the recombinant protein LamB after emulsification new zealand white rabbit.Before the immunity, give over to negative serum by rabbit ear edge arterial blood extracting.During first immunisation, antigen and isopyknic Freund's complete adjuvant emulsification, the subcutaneous multi-point injection in back, immunizing dose is about 250 μ g albumen/kg rabbits.Every two all booster immunizations once, during booster immunization, antigen and Freund's incomplete adjuvant emulsification.For the second time behind the booster immunization the 8th day by ear edge arterial blood extracting 1mL, measure serum titer.Use the Dot-ELISA method to survey it and tire, reaching satisfied tiring after, the carotid artery bloodletting.Centrifugation serum, tubule packing, freezing preservation.
The cross reaction of recombinant protein rabbit anti-serum and vibrios outer membrane protein: with the antiserum(antisera) of recombinant protein LamB as primary antibodie, bacterial protein with 18 strain Protein Profiles of Vibrio Sps and the non-vibrios of 3 strains reacts respectively, carry out Western blotting and analyze, the results are shown in Figure 3.
The cross immunity protectiveness of restructuring maltoporin LamB take zebra fish as model evaluation.
Zebra fish is raised: the tap water with abundant aeration is raised, and keeps water temperature about 26 ± 2 ℃, is living under the oxygenation condition more than 1 week to conform.Change water once early morning every day, and the morning and afternoon respectively feed and raise once, and the feeding amount is eaten up with 5min and is advisable.
The calculating of bacterial strain LD50: cultivate after pathogenic vibrio (Vibrio parahaemolyticus VPL49-0, Vibrio parahaemolyticus, vibrio alginolyticus, Vibrio mimicus, Vibrio harveyi, the Vibrio furnissii) activation, count and with fish with normal saline dilution to 10
9CFU/mL, then ten times of gradient dilutions to 10
6CFU/mL.Every group of 10 zebra fishs, every zebra fish of experimental group are injected 10 μ L bacterium liquid, and control group is injected 10 μ L physiological saline.Calculate vibrios to the medium lethal dose LD50 of zebra fish with reference to the method for Reed-Mueneh.Calculation formula is as follows:
LgLD50=lg is higher than the bacterium extent of dilution+distance proportion of 50% mortality ratio * lg dilution factor
Distance proportion=(being higher than 50% mortality ratio-50%)/(be higher than 50% mortality ratio-be lower than 50% mortality ratio)
The experiment of recombinant protein rabbit anti-serum passive protection: healthy zebra fish is randomized 30 every group.The rabbit anti-serum of the zebra fish abdominal injection recombinant protein LamB of experimental group, injection volume is every of 10 μ L/, the physiological saline of control group injection equal volume amounts.Behind the 2h, adopt pathogenic vibrio (vibrio alginolyticus ATCC33787, Vibrio parahaemolyticus ATCC17802, Vibrio parahaemolyticus VPL4-90 and Vibrio mimicus ATCC33653) the bacterium liquid of 500 times of LD50 to attack poison, the survival condition of observation experiment group and control group zebra fish records the number of dead fish in 5 days.Calculate relative survival rate RPS:
Relative survival rate (RPS)=(the dead fish number of the 1-immune group/dead fish number of control group) * 100%
Found that the rabbit anti-serum of recombinant protein has the passive immunization provide protection to many strains vibrios, experimental result sees Table 1.
Table 1
The active immunity of zebra fish experiment: first immunisation, the recombinant protein LamB antigen of the 2mg/ml that every zebra fish abdominal injection 5 μ L are obtained by embodiment 5.After the first immunisation, carry out week about booster immunization one time.For the second time booster immunization is selected 3-5 bar zebra fish after two weeks at random, collects blood, detects the situation that produces antibody in the fish body with the dot-ELISA method, as reaches ideal and tire and can attack the poison experiment.Substitute recombinant protein LamB antigen with physiological saline, injected dose and the identical control group that is used as of operation.
Attack poison: the vibrios behind the multiple poison of activation is cultured to logarithmic phase.Centrifugal collection thalline washs 3 times with physiological saline with the fish of sterilization, removes poisonous composition in the substratum.Use at last physiological saline resuspended, carry out bacterial count.Adopt colony counting method and counting method of blood cell to count, and adjust bacterial concentration with physiological saline, bacterium liquid (vibrio alginolyticus ATCC33787, Vibrio parahaemolyticus ATCC17802, Vibrio parahaemolyticus VPL4-90, Vibrio mimicus ATCC33653, Vibrio harveyi NBRC15634 and Vibrio furnissii ATCC33813) injection zebra fish with 500 times of LD50 is attacked poison, and observe and attack growth and the pathology situation of respectively organizing the fish body in malicious rear 5 days, record the number of dead fish.Calculate relative survival rate RPS, comprehensive fish pathology damage is analyzed, thereby estimates the effect of vaccine, and it has the cross neutralization protectiveness to Vibrio, and experimental result sees Table 2.
Table 2
Although, above used general explanation, embodiment and test, the present invention is described in detail, on basis of the present invention, can make some modifications or improvements it, and this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. maltoporin encoding gene LamB, it is characterized in that this gene has the nucleotide sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8.
2. preparation method of maltoporin LamB that recombinates, the method comprises the steps:
(1) clone of maltoporin encoding gene LamB;
(2) structure of coli expression carrier pET32-LamB;
(3) structure of intestinal bacteria recombinant bacterium BL21-pET32-LamB;
(4) preparation of restructuring maltoporin LamB.
3. method according to claim 2 is characterized in that, the method comprises the steps:
(1) clone of maltoporin encoding gene LamB: the design degenerated primer, take the genomic dna of vibrios as template, carry out PCR, obtain maltoporin encoding gene LamB; Then be connected among the pMD19-T, make up escherichia coli cloning carrier pMD19-T-LamB; This carrier is converted into DH5 α competent cell, makes up intestinal bacteria recombinant bacterium DH5 α-pMD19-T-LamB, order-checking; Wherein the degenerated primer sequence is as follows:
LamB-F:5'-ATGAAAAAAGTAAGTSNYATTGCAG-3'
LamB-R:5'-TTACCACCAAGCTTCNRCTTG-3'
Wherein, S=C/G; N=A/C/G/T; Y=C/T; R=A/G;
(2) structure of coli expression carrier pET32-LamB: primer is expressed in design, take cloning vector pMD19-T-LamB as template, carry out PCR, the LamB gene and the expression vector pET32a (+) that obtain are carried out double digestion with BamH I and Xho I, connect, make up coli expression carrier pET32-LamB; It is as follows wherein to express primer sequence:
LamBeF:5'-CGCGGATCCGCAGTGGATTTTAAC-3'
LamBeR:5'-CCGCTCGAGATTCGGCTTTTACCAC-3'；
(3) structure of intestinal bacteria recombinant bacterium BL21-pET32-LamB: the coli expression carrier pET32-LamB in (2) is transformed the BL21 competent cell, obtain intestinal bacteria recombinant bacterium BL21-pET32-LamB;
(4) preparation of restructuring maltoporin LamB: contain the penbritin of 100 μ g/ml at liquid LB() access intestinal bacteria recombinant bacterium BL21-pET32-LamB bacterium liquid in (3) of overnight incubation in the substratum by 1% ratio, add 1mM isopropylthio-β-D-galactoside (IPTG) at 37 ℃ after being cultured to OD600=0.6, carry out abduction delivering 6h in 37 ℃ of temperature; Collect thalline, purifying inclusion body, inclusion body purification liquid are directly crossed nickel NTA sepharose FF post, the affinitive layer purification recombinase, and imidazoles gradient competitive elution obtains the restructuring maltoporin LamB of purifying.
4. the restructuring maltoporin LamB that is prepared by claim 2 or 3 described methods.
5. the coli expression carrier pET32-LamB for preparing restructuring maltoporin LamB claimed in claim 4.
6. the intestinal bacteria recombinant bacterium BL21-pET32-LamB for preparing restructuring maltoporin LamB claimed in claim 4.
7. a vibrios intersecting protective vaccine is characterized in that, this vaccine contains restructuring maltoporin LamB claimed in claim 4.
8. the medicine for the treatment of vibriosis is characterized in that this medicine contains restructuring maltoporin LamB claimed in claim 4.
9. the antibody for preparing take restructuring maltoporin LamB claimed in claim 4 as immunogen.
10. the diagnostic kit for detection of vibriosis that contains the described antibody of claim 9.
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