CN105968212A - Eel Vibrio vulnificus/Edwardsiella tarda duplex recombinant protein and preparation method thereof - Google Patents
Eel Vibrio vulnificus/Edwardsiella tarda duplex recombinant protein and preparation method thereof Download PDFInfo
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- CN105968212A CN105968212A CN201610351570.4A CN201610351570A CN105968212A CN 105968212 A CN105968212 A CN 105968212A CN 201610351570 A CN201610351570 A CN 201610351570A CN 105968212 A CN105968212 A CN 105968212A
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- vibrio vulnificus
- outer membrane
- membrane protein
- tarda
- bigeminal
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- 241000607471 Edwardsiella tarda Species 0.000 title claims abstract description 35
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- 108010008281 Recombinant Fusion Proteins Proteins 0.000 title claims abstract description 30
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- Biomedical Technology (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The invention discloses an eel Vibrio vulnificus/Edwardsiella tarda duplex recombinant protein and a preparation method thereof. The duplex recombinant protein comprises a Vibrio vulnificus outer membrane protein OmpU extramembrane part and an Edwardsiella tarda outer membrane protein OmpA extramembrane part which are connected together, wherein the Vibrio vulnificus outer membrane protein OmpU extramembrane part and Edwardsiella tarda outer membrane protein OmpA extramembrane part respectively comprise 238th-456th amino acid sequences and 467th-695th amino acid sequences in SEQ ID NO.01. The Vibrio vulnificus outer membrane protein OmpU extramembrane part and Edwardsiella tarda outer membrane protein OmpA extramembrane part are connected together by a gene engineering means to establish the duplex recombinant protein. After being used for immunizing the eel, the protein can simultaneously resist the infection of Vibrio vulnificus and Edwardsiella tarda. Compared with the duplex inactivated vaccine, the duplex recombinant protein has higher relative immunity protection effect and lower toxic and side effects.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of anguilla japonica Vibrio vulnificus and edwardsiella tarda bigeminal restructuring
Albumen and preparation method thereof.
Background technology
Over nearly 20 years, there is fulminant septicaemia in China's cultured freshwater fish, has carried out immeasurable economy to cultivation industrial belt
Loss, research shows that the main pathogenic fungi of this disease is Vibrio vulnificus and Edwardsiella tarda of pausing late.Vibrio vulnificus
(Aeromonas hydrophila) and Edwardsiella tarda (Edwardsiella tarda) they are the important pathogen of anguilla japonica,
Spring and summer alternately time the energy-conservation disease causing the infectiousness such as anguilla japonica hueppe's disease, Liver and kidney enlargement, gill rot, tail-rot disease the strongest,
America anguilla japonica, European eel and the Japanese eel of artificial cultivation all can occur.Culture fishery life-time service chemotherapy
This disease, owing to producing the reasons such as drug resistance, causes available safe drugs fewer and feweri.Recently visible about Vibrio vulnificus and
The research that Edwardsiella tarda full bacterium inactivated vaccine is relevant with subunit vaccine, but to apply these achievements in research that anguilla japonica is carried out
Immunity, to prevent the infringement of multiple pathogen, needs anguilla japonica is carried out multiple injection operation, relatively big to the injury of fish body, and operation
Loaded down with trivial details.
Summary of the invention
It is an object of the invention to overcome prior art defect, it is provided that a kind of anguilla japonica Vibrio vulnificus that can use as vaccine is with late
Blunt tarda bigeminal recombinant protein.
Another object of the present invention is to provide the preparation method of this bigeminal recombinant protein.
One technical scheme of the present invention is:
A kind of anguilla japonica Vibrio vulnificus and edwardsiella tarda bigeminal recombinant protein, including the Vibrio vulnificus adventitia linked together
Albumen OmpU film outer portion and late a tarda outer membrane protein OmpA film outer portion, its aminoacid sequence wraps respectively
Include the aminoacid sequence shown in the 238th～456 and the 467th～695 of SEQ ID NO 01.
In a preferred embodiment of the invention, described Vibrio vulnificus outer membrane protein OmpU film outer portion and late a love
Moral Fahrenheit bacterial outer membrane protein OmpA film outer portion is linked together by one section of connection peptides, and the sequence of this connection peptides includes SEQ
The aminoacid sequence shown in 457th～466 of ID NO 01.
The present invention another solution is that
A kind of encode described anguilla japonica Vibrio vulnificus and the gene order of edwardsiella tarda bigeminal recombinant protein, including connecting
The coding nucleotide sequence of Vibrio vulnificus outer membrane protein OmpU film outer portion together and late a tarda adventitia egg
The coding nucleotide sequence of white OmpA film outer portion, above-mentioned coding nucleotide sequence includes the of SEQ ID NO 02 respectively
712～1368 and the 1399th～2085 shown nucleotide sequence.
In a preferred embodiment of the invention, the gene sequence of described Vibrio vulnificus outer membrane protein OmpU film outer portion
The gene order of row and late a tarda outer membrane protein OmpA film outer portion includes SEQ ID NO's 02 by one section
The nucleotide sequence of the 1369th～1398 shown nucleotide sequence coded one section of connection peptides couples together.
The yet another aspect of the present invention is:
A kind of express described anguilla japonica Vibrio vulnificus and the plasmid of edwardsiella tarda bigeminal recombinant protein, including being loaded with bag
Include the prokaryotic expression carrier of nucleotide sequence shown in SEQ ID NO 02.
In a preferred embodiment of the invention, described prokaryotic expression carrier is fusion expression vector.
The yet another aspect of the present invention is:
A kind of express described anguilla japonica Vibrio vulnificus and the escherichia coli of edwardsiella tarda bigeminal recombinant protein, this large intestine bar
Bacterium converts has load to include the prokaryotic expression carrier of nucleotide sequence shown in SEQ ID NO 02.
In a preferred embodiment of the invention, described prokaryotic expression carrier is fusion expression vector.
In a preferred embodiment of the invention, described prokaryotic expression carrier be pGEX-2T-His or
pGEX-2TH-his。
In a preferred embodiment of the invention, the described escherichia coli for expressing are e. coli bl21.
The yet another aspect of the present invention is:
A kind of above-mentioned anguilla japonica Vibrio vulnificus and the preparation method of edwardsiella tarda bigeminal recombinant protein, comprise the steps:
(1) amplification coding Vibrio vulnificus outer membrane protein OmpU film outer portion and late a tarda outer membrane protein respectively
The gene order of OmpA film outer portion, its aminoacid sequence includes the 238th～456 and of SEQ ID NO 01 respectively
Aminoacid sequence shown in 467～695, its nucleotide sequence is respectively such as the 831st～1612 and of SEQ ID NO 02
Shown in 1399～2085；
(2) two gene orders of step (1) gained are linked together, obtain fusion gene sequence；
(3) the fusion gene sequence of step (2) gained is connected in prokaryotic expression carrier, builds recombinant expression carrier；
(4) recombinant expression carrier constructed in step (3) is transformed in prokaryotic host cell, and is being suitable to expression
The prokaryotic host cell being converted is cultivated under conditions of described fusion gene；
(5) fusion protein that the prokaryotic host cell that also purification is cultivated is expressed is reclaimed；
(6) fusion protein of step (5) gained is carried out renaturation.
The invention has the beneficial effects as follows:
1, the present invention is by the outer membrane protein OmpU film outer portion of Vibrio vulnificus and a tarda outer membrane protein OmpA late
Film outer portion is linked together structure bigeminal recombinant protein by genetic engineering means, after this protein immunization anguilla japonica, can prevent simultaneously
Imperial Vibrio vulnificus and the infection of Edwardsiella tarda, the immunity comparing single outer membrane protein has more preferable immune effect；
2, the bigeminal recombinant protein of the present invention only needs fish body is carried out a shot, can obtain the immunity to two kinds of pathogen
Effect, decreases the injury to fish body, simplifies immunity step；
3, the preparation method of the bigeminal recombinant protein of the present invention is suitable for industrialized production.
Accompanying drawing explanation
Fig. 1 is anguilla japonica Survival curves figure after 28d Vibrio vulnificus counteracting toxic substances after immunity in the embodiment of the present invention 2.
Fig. 2 is that in the embodiment of the present invention 2, after immunity, 28d pauses the anguilla japonica Survival curves figure after tarda counteracting toxic substances late.
Fig. 3 is the ELISA titer change of anguilla japonica different times serum specific antibody after immunity in the embodiment of the present invention 2
Figure.
Fig. 4 be in the embodiment of the present invention 2 anguilla japonica after immunity at serum, mucus, liver organization and renal tissue suspension not
The activity change figure of lysozyme of the same period.
Detailed description of the invention
Combine accompanying drawing below by way of detailed description of the invention technical scheme is further detailed and describes.
Embodiment 1
The present embodiment carries out anguilla japonica Vibrio vulnificus and the edwardsiella tarda bigeminal recombinant protein of the present invention.
(OMP-Vibrio-Edwa) preparation
(1) in the Vibrio vulnificus outer membrane protein ompU chosen and Edwardsiella tarda outer membrane protein ompA gene outside film
Region immunogenicity and hydrophilic all preferably DNA fragmentation, couples together with joint in the middle of choose two fragments, if
The upstream and downstream primer of meter amplification OmpU Gene Partial fragment is respectively as follows: P1:5'CCG GAA TTC TAC GCA
GGT CTA GGC GGC AAG T 3'(SEQ ID NO 03,31bp, introduces EcoR I site)；P2:5'ACC CGA
GCC ACC ACC GCC CGAGCC TAT ACG AGC GTA GCC AGC ACC GCC AAC T 3'(SEQ
ID NO 04,52bp)；The upstream and downstream primer difference of amplification OmpA portion gene: P3:5'TCG GGC GGT GGC
GGC TCG GGT GGC GGA TCA GTA TGG CGT TCT GAT ATC CAC GG3 ' (SEQ ID NO 05,
53bp)；P4:5'CGC GTC GAC CTG CGG CTG AGA AAC TTC TTC TT 3'(SEQ ID NO 06,
32bp, introduces Sal I site).Above-mentioned primer is synthesized by Shanghai JaRa bio-engineering corporation, wherein primer P 25 " holds
21 bases (3't caa ccg cca cga ccg atg cga gca tat ccg agc ccg cca cca ccg agc cca) 5' and P3 5'
Hold 21 bases (5'tcg ggc ggt ggc ggc tcg ggt ggc gga tca gta tgg cgt tct gat atc cac gg3 ') complementary, in
Between joint altogether 30bp.(cgtataggctcgggcggtggtggctcgggt).
Using the genomic DNA of Vibrio vulnificus as PCR reaction template, with P1 and P2 as primer, expand OmpU
Genetic fragment, reclaims PCR primer；Use following system (50 μ L) amplification gene fragment:
PCR reaction condition is as follows: 94 DEG C of denaturations 5min；94 DEG C of degeneration 1min, 54 DEG C of Gradient annealing 1min are (every
Individual gradient increase was once), 72 DEG C extend 2min, 30 circulations；72 DEG C extend 10min；4 DEG C of preservations.Use 1%
After sepharose electrophoresis inspection PCR primer, find that optimum annealing temperature is 56 DEG C, run glue rubber tapping the most in a large number and reclaim wound
Vibrio OmpU genetic fragment, uses glue to reclaim test kit and reclaims, and use NanoDrop 2000 ultramicron light splitting
Nucleic acid concentration after photometer detection recovery.
Using the genomic DNA of Edwardsiella tarda as PCR reaction template, with P3 and P4 as primer, amplification
OmpA genetic fragment, reclaims PCR primer, and PCR reaction system is as follows:
PCR reaction condition is as follows: 94 DEG C of denaturations 5min；94 DEG C of degeneration 1min, 54 DEG C of Gradient annealing 1min are (every
Individual gradient increase was once), 72 DEG C extend 2min, 30 circulations；72 DEG C extend 10min；4 DEG C of preservations.Use 1%
After sepharose electrophoresis inspection PCR primer, finding that optimum annealing temperature is 58 DEG C, then after PCR amplification, glue recovery is blunt
Tarda OmpA genetic fragment, and use the detection of NanoDrop 2000 ultramicrospectrophotometer to reclaim nucleic acid
Concentration.
(2) by the outer membrane protein OmpU film outer portion of the Vibrio vulnificus of above-mentioned gained and the tarda outer membrane protein that pauses late
The nucleotide sequence of OmpA film outer portion links together, and obtains fusion gene sequence, and method of attachment is specific as follows:
The aim sequence glue that amplification is arrived by a reclaims, the same E.Z.N.A. of methodTMGel Extraction Kit operation instructions, and
Measure its concentration.
B uses two-step method to connect the DNA genetic fragment that above-mentioned two glue reclaims: use grads PCR method to determine optimal annealing
Temperature, condition and system are as follows:
The first step, PCR reaction condition: 94 DEG C of denaturations 5min；94 DEG C of degeneration 45s, 56 DEG C of annealing 45s, 72 DEG C
Extend 4min, 13 circulations；4 DEG C of preservations.Reaction system:
Second step, PCR reaction condition: 94 DEG C of denaturations 5min；94 DEG C of degeneration 45s, 56.3 DEG C of annealing 45s, 72 DEG C
Extend 4min, 13 circulations；72 DEG C of extensions, 4 DEG C of preservations.Reaction system:
After having reacted, purified pcr product, obtain PCR primer G-Vibrio-Edwa and (include SEQ ID NO 02
Shown sequence)；
(3) PCR primer G-Vibrio-Edwa obtained is added polyA tail, uses tailing test kit to carry out tailing,
Then reclaim test kit with glue to reclaim, it is thus achieved that with the purpose fragment of polyA tail, by this purpose fragment and amalgamation and expression
Vector pGEX-2TH-his (purchased from GE healthcare) all connects at T4DNA with after EcoR I and Sal I double digestion
It is attached obtaining recombinant expression carrier under the effect of enzyme；
(4) recombinant expression carrier is transformed in e. coli bl21 competent cell, obtains recombinant bacterium BL21；With the denseest
The IPTG of degree 0.25mM～2.0mM induces recombinant bacterium BL21 to express recombiant protein at a temperature of 16～37 DEG C
(OMP-Vibrio-Edwa) 1.5～9 hours, wherein bacterium solution initial concentration was OD600Nm=0.1～1.0.
(5) collect the thalline of the recombinant bacterium BL21 after induction, carry out ultrasonic disruption, centrifugal (4 DEG C, 10000rpm,
After 5min) collecting washing precipitation, by the resuspended precipitation of the PBS of pre-cooling also, after similarity condition is centrifugal, takes supernatant and carry out pure
Change；Carry out above-mentioned supernatant nickel post tying purification, obtain fusion protein OMP-Vibrio-Edwa；
(6) the OMP-Vibrio-Edwa dialysis of eluting being carried out renaturation, renaturation carries out lyophilizing i.e. get Ben Fa after completing
Bright anguilla japonica Vibrio vulnificus and edwardsiella tarda bigeminal recombinant protein (including the sequence shown in SEQ ID NO 01).
Embodiment 2
(1) by PBS blank, Vibrio vulnificus and edwardsiella tarda bigeminal inactivated bacterial liquid and embodiment 1 gained
The anguilla japonica Vibrio vulnificus of the present invention and edwardsiella tarda bigeminal recombinant protein respectively by the mode of lumbar injection to three groups
Anguilla japonica (50g/ tail) carries out immunity, and often the most corresponding PBS control group (PBS Group) of group anguilla japonica, bigeminy inactivated bacteria are exempted from
Epidemic disease group (Bivalent FKC Group) and double expression(DE) outer membrane protein immune group (Bivalent OMP Group), wherein this
Bright anguilla japonica Vibrio vulnificus and edwardsiella tarda bigeminal recombinant protein are before the test with the PBS (pH=7.4) of 0.01M
It is configured to 1mg/mL, it is fully mixed with the incomplete Freund's adjuvant of equivalent, final concentration of 0.5mg/mL；Wound arc
Bacterium and late a tarda, after 28 DEG C and 32 DEG C of pancreas peptone soybean broths (TSB) cultivate 24h, use 0.01M
PBS (pH=7.4) be made into 5.0 × 10 respectively8Bacterium solution (the OD of cfu/mL595nm=0.5) equivalent mixing afterwards, in mixing
Liquid adds the incomplete Freund's adjuvant with thalline equivalent and the formalin of final concentration of 0.4%, takes after 4 DEG C of standing 24h
A small amount of bacterium solution coats tryptose soya agar (TSA), is Vibrio vulnificus with blunt when cultivating 24h without colony growth
Tarda bigeminal inactivated bacterial liquid；PBS blank is that 0.01M PBS (pH=7.4) is the completeest with the Freund of above-mentioned equivalent
The mixing of full adjuvant；The amount of liquid of every tail anguilla japonica lumbar injection is 0.2mL；
The acquisition method of laboratory sample is particularly as follows: anguilla japonica 14d, 21d, 28d and 42d eugenol (100 after immunity
Ppm) anesthesia blood sampling, often organizes and takes 5 tail anguilla japonica blood every time.Every tail anguilla japonica about 0.5mL blood heparin sodium of 20 μ L resists
Solidifying (0.5UI/ μ L) latter 4 DEG C stands overnight, draws upper plasma and measure its antibody titer and lysozyme content, concurrently separate not
Carry out cultivating and transformation experiment with cell component.Before blood sampling, every tail anguilla japonica takes mucus 0.1g, takes liver and kidney after blood sampling
Organizing each 0.1 gram, above-mentioned mucus and tissue all add the PBS (pH=7.4) of 0.5mL 0.01M and carry out homogenate with ultrasonic
Processing, take supernatant after 10000rpm is centrifugal ,-70 DEG C save backup
(2) 28d after above-mentioned three groups of anguilla japonica immunity, uses Vibrio vulnificus (6 × 108Cfu/ tail) and the tarda (2 × 10 that pauses late6
Cfu/ tail) carry out counteracting toxic substances infection respectively.As it is shown in figure 1, PBS control group, the immunity of bigeminy inactivated bacteria after contaminated wound vibrio
The anguilla japonica of group and double expression(DE) outer membrane protein immune group mortality rate in 14d is respectively 50%, 10% and 0%；With PBS pair
Comparing according to group, the relative immunity protective rate of anguilla japonica is respectively by bigeminy inactivated bacteria immune group and double expression(DE) outer membrane protein immune group
80% and 100%, X 2 test result shows, after counteracting toxic substances after 5d to 14d, double expression(DE) outer membrane protein immune group
Survival rate significantly improves (p < 0.05) compared with matched group, but the difference of PBS control group and bigeminy inactivated bacteria immune group is being attacked
Difference after poison 14d is not up to significant level (p > 0.05)；As in figure 2 it is shown, after counteracting toxic substances infects the tarda that pauses late,
The anguilla japonica of PBS control group, bigeminy inactivated bacteria immune group and double expression(DE) outer membrane protein immune group mortality rate in 14d is respectively
It is 60%, 30% and 10%；Compared with PBS control group, bigeminy inactivated bacteria immune group and double expression(DE) outer membrane protein immune group
The relative immunity protective rate of anguilla japonica is respectively 50% and 83%.X 2 test result shows, after counteracting toxic substances infection, 12d is extremely
14d, the relative immunity protective rate of double expression(DE) outer membrane protein immune group notable (p < 0.05) is higher than matched group, and PBS control
The difference of group and bigeminy inactivated bacteria immune group difference after counteracting toxic substances 14d is not up to significant level (p > 0.05).
As it is shown on figure 3, anguilla japonica is after immunity processes, bigeminy inactivated bacteria immune group is relative with double expression(DE) outer membrane protein immune group
PBS control group (14,21,28 and 42d) in experiment periods is respectively provided with higher antibody response, but except 42d (##P<
0.01), beyond, the antibody titer between double expression(DE) outer membrane protein immune group and bigeminy inactivated bacteria immune group is all without marked difference.
From 14d to 28d, log 2 titre of double expression(DE) outer membrane protein immune group and bigeminy inactivated bacteria immune group all reaches more than 6.0,
It is significantly higher than PBS control group.The titre of double expression(DE) outer membrane protein immune group is continued for improving to 42d from 14d, but two
The titre of connection inactivated bacteria immune group is on a declining curve from 28d to 42d, and the antibody titer of PBS control group is from 28d to 42d
Present growth, but its difference is the most notable.
As shown in Figure 4,14d after immunity, the serum lysozyme activities reached of bigeminy inactivated bacteria immune group notable (* P < 0.05) is low
In matched group, and 21d after immunity, the serum lysozyme activities reached of double expression(DE) outer membrane protein immune group notable (* P < 0.05) is high
In other two groups, to 28d and 42d, the serum lysozyme activities reached in three groups is reduced to phase same level (Fig. 4 A)；After immunity
14 to 42d, during except 21d, there is significance (P < 0.05) difference with other two groups in double expression(DE) outer membrane protein immune group,
Remaining time, the mucus lysozyme activity of three groups is maintained at phase same level (Fig. 4 B)；21d after immunity, bigeminy inactivated bacteria is exempted from
The liver lysozyme activity of epidemic disease group notable (P < 0.01) is higher than other two groups, 28d after immunity, double expression(DE) external mold protein immunization
There is significance (P < 0.01) difference, between bigeminy inactivated bacteria immune group and PBS control group between group and PBS control group
There is significance (P < 0.05) difference (Fig. 4 C)；Compared with PBS control group, the kidney of bigeminy inactivated bacteria immune group is molten
Bacterium enzymatic activity 14d after immunity compares PBS control group significance (P < 0.01) to be increased, double expression(DE) outer membrane protein immune group
Kidney lysozyme activity 28d after immunity compare PBS control group significance (P < 0.01) increase, double expression(DE) adventitia egg
There is significant difference (P < 0.01) in kidney lysozyme activity 28d after immunity of white immune group and bigeminy inactivated bacteria immune group
(Fig. 4 D).
The above, only presently preferred embodiments of the present invention, therefore the scope that the present invention implements can not be limited according to this, i.e. depend on
The equivalence change that the scope of the claims of the present invention and description are made with modify, all should still belong in the range of the present invention contains.
Claims (10)
1. an anguilla japonica Vibrio vulnificus and edwardsiella tarda bigeminal recombinant protein, it is characterised in that: include being connected to one
The Vibrio vulnificus outer membrane protein OmpU film outer portion risen and the tarda outer membrane protein OmpA film outer portion that pauses late, its
Aminoacid sequence includes the aminoacid sequence shown in the 238th～456 and the 467th～695 of SEQ ID NO 01 respectively.
2. a kind of anguilla japonica Vibrio vulnificus as claimed in claim 1 and edwardsiella tarda bigeminal recombinant protein, its feature
It is: outside described Vibrio vulnificus outer membrane protein OmpU film outer portion and late a tarda outer membrane protein OmpA film
Dividing and linked together by one section of connection peptides, the sequence of this connection peptides includes shown in the 457th～466 of SEQ ID NO 01 the
Aminoacid sequence.
3. one kind encodes described in claim 1 anguilla japonica Vibrio vulnificus and the gene of edwardsiella tarda bigeminal recombinant protein
Sequence, it is characterised in that: include the coding nucleotide of the Vibrio vulnificus outer membrane protein OmpU film outer portion linked together
The coding nucleotide sequence of sequence and late a tarda outer membrane protein OmpA film outer portion, above-mentioned coding nucleotide sequence
Row include the 712nd～1368 and the 1399th～2085 the shown nucleotide sequence of SEQ ID NO 02 respectively.
A kind of base encoding anguilla japonica Vibrio vulnificus and edwardsiella tarda bigeminal recombinant protein the most as claimed in claim 3
Because of sequence, it is characterised in that: the coding nucleotide sequence of described Vibrio vulnificus outer membrane protein OmpU film outer portion and slow
The coding nucleotide sequence of tarda outer membrane protein OmpA film outer portion includes the of SEQ ID NO 02 by one section
The nucleotide sequence of 1369～1398 shown nucleotide sequence coded one section of connection peptides couples together.
5. one kind is expressed described in claim 1 anguilla japonica Vibrio vulnificus and the matter of edwardsiella tarda bigeminal recombinant protein
Grain, it is characterised in that: include being loaded with including the prokaryotic expression carrier of nucleotide sequence shown in SEQ ID NO 02.
A kind of matter expressing anguilla japonica Vibrio vulnificus and edwardsiella tarda bigeminal recombinant protein the most as claimed in claim 5
Grain, it is characterised in that: described prokaryotic expression carrier is fusion expression vector.
7. one kind is expressed described in claim 1 anguilla japonica Vibrio vulnificus and the large intestine of edwardsiella tarda bigeminal recombinant protein
Bacillus, it is characterised in that: described escherichia coli convert has load to include the protokaryon of nucleotide sequence shown in SEQ ID NO 02
Expression vector.
The most as claimed in claim 7 a kind of express the big of anguilla japonica Vibrio vulnificus and edwardsiella tarda bigeminal recombinant protein
Enterobacteria, it is characterised in that: described prokaryotic expression carrier is fusion expression vector.
9. a kind of expression anguilla japonica Vibrio vulnificus and edwardsiella tarda bigeminal recombinant protein
Escherichia coli, it is characterised in that: described for express escherichia coli be e. coli bl21.
10. the anguilla japonica Vibrio vulnificus described in a claim 1 and the preparation side of edwardsiella tarda bigeminal recombinant protein
Method, it is characterised in that: comprise the steps:
(1) amplification coding Vibrio vulnificus outer membrane protein OmpU film outer portion and late a tarda outer membrane protein respectively
The gene order of OmpA film outer portion, its nucleotide sequence is respectively such as the 712nd～1368 and of SEQ ID NO 02
Shown in 1399～2085；
(2) two gene orders of step (1) gained are linked together, obtain fusion gene sequence；
(3) the fusion gene sequence of step (2) gained is connected in prokaryotic expression carrier, builds recombinant expression carrier；
(4) recombinant expression carrier constructed in step (3) is transformed in prokaryotic host cell, and is being suitable to expression
The prokaryotic host cell being converted is cultivated under conditions of described fusion gene；
(5) fusion protein that the prokaryotic host cell that also purification is cultivated is expressed is reclaimed；
(6) fusion protein of step (5) gained is carried out renaturation.
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