CN100500850C - V. alginolyticus outer-membrane protein W gene, process for preparing the same and uses thereof - Google Patents
V. alginolyticus outer-membrane protein W gene, process for preparing the same and uses thereof Download PDFInfo
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Abstract
The invention relates to a V. alginolyticus outer-membrane protein W gene, process for preparing the same and uses. The invention provides a novel V.alginolyticus OmpW gene, and the use of V.alginolyticus and Vibrio vulnficus OmpW protein and coded sequence, which comprises, linking purified nucleic acid sequence encoding protein active polypeptides having V.alginolyticus OmpW to expression regulation sequence, forming V.alginolyticus OmpW protein expression carrier, transferring the expression carrier into host cells, forming the recombination cells of the V.alginolyticus Omp protein, culturing the recombination cells and separating.
Description
Technical field
The present invention relates to a kind of gene, especially relate to a kind of outer membrane protein W (outer membrane protein W, OmpW) gene and preparation method and its usage of on vibrio alginolyticus (Vibrio alginolocius) adventitia, expressing.
Background technology
Vibrios is the important ocean pathogenic bacteria of a class, is the main pathogenic former of gastrointestinal tract disease in the coastland, and they also are the important pathogenic bacterias in the culture fishery simultaneously, and the generation of vibriosis often causes enormous economic loss.Adaptive faculty to different salinity environment is a kind of basic function of ocean pathogenic microbes, this also is closely related with bacterium pathogenecity and breeding transmission capacity simultaneously, and outer membrane protein is in the outermost layer of bacterium, directly contact with external environment (comprising ocean environment and host's internal milieu two sides), it is launched further investigation is an important aspect analyzing bacterium salinity adaptation mechanism.In sea water culture environment, the Vibrio bacterium accounts for very big proportion in each bacterioid, accounts for clear superiority with vibrio alginolyticus (V.alginolyticus), Vibrio anguillarum (V.anguillarium) and Vibrio parahaemolyticus (V.parahaemolyticus) especially.Because it extensively is present in natural waters, has become sea farming and endangered one of severe diseases as infecting pathogeny spread and epidemic disease.Vibrio alginolyticus is to be named by Miyamoto in 1961, because biological characteristics has many places similar to Vibrio parahaemolyticus, belong to the living vibrios in halophilism sea together, so " classified it as Vibrio parahaemolyticus biological II type bacterium { Vibrio parahaemolyticus is biological I type } in the uncle Jie Shi Bacteria Identification handbook in 1974, G+C content difference among the DNA of back confirmation two bacterium, " uncle Jie Shi system Bacteria Identification handbook just marks it to come version in 1984 from Vibrio parahaemolyticus, independently be a kind, has caused everybody concern.Report successively both at home and abroad for many years from diarrhoea and food poisoning patient ight soil and detect this bacterium.OmpW is an important albumen in the bacterial outer membrane albumen, at present Vibrio parahaemolyticus (The Lacent only in vibrios
Volume 361, and Issue 93591 March 2003, Pages743-749 Genome sequence of Vibrio parahaemolyticus:a pathogenic mechanism distinct fromthat of V cholerae) and vibrio cholerae (Nucleic Acids Res.1990 April 25; 18 (8): 2180.Nucleotidesequence of the gene, ompW, encoding a 22kDa immunogenic outer membrane protein of Vibriocholerae.) measured the nucleotide sequence of this gene, and its function yet there are no elaboration; In intestinal bacteria, also contain the OmpW gene, think that at present this albumen is a polarity albumen (Mol Microbiol 2004 May; 52 (4) 129-44 Proteomicscreening and identification of differentially distributed membrane proteins in Escherichia coli), and be or that the acceptor of colicin is main member (J Bacteriol 1999 Jun 181 (11): 3578-81Characterization of colicin S4 and its receptor of acceptor, OmpW, a minor protein of the Escherichia coliouter membrane).Shang Weijian has any molten alginic acid OmpW nucleotide sequence of mentioning that discloses or reported.Do not see yet and reported that vibrios OmpW albumen had the salt tolerant function.
Summary of the invention
The object of the present invention is to provide a kind of new vibrio alginolyticus OmpW gene, it is the nucleotide sequence that coding has the polypeptide of vibrio alginolyticus OmpW protein active.
Another object of the present invention provides a kind of new proteic aminoacid sequence of vibrio alginolyticus OmpW.
The 3rd purpose of the present invention provides a kind of method of utilizing recombinant technology to prepare above-mentioned new vibrio alginolyticus OmpW gene.
The present invention also provides the application of this vibrio alginolyticus and Vibrio parahaemolyticus OmpW albumen and encoding sequence, and this proteic a kind of new function, i.e. salt tolerant function promptly is provided.
The said vibrio alginolyticus OmpW of the present invention gene is the nucleotide sequence that coding has the polypeptide of vibrio alginolyticus OmpW protein active, is designated as SEQ ID NO.3, promptly
(i) sequence signature:
(A) length: 642bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: Nucleotide
(iii). sequence description: SEQ ID NO.3
1 ATGAAAAAAA CAATCTGCAG TCTAGCAGTG GTTGCTGCAC TCGTGTCACC AAGTGTTTTC
61 GCTCATAAAC AGGGTGACTT CGTTCTTCGT GTTGGTGCGG CGTCTGTCGT TCCAAATGAC
121 AGCAGTGATA AGATTCTTGG TTCTCAAGAA GAGTTAGAAG TTGACTCAAA TACGCAGCTT
181 GGTTTGACGT TTGGCTACAT GTTCACAGAC AACATCAGTT TAGAGCTTCT AGCAGCAACA
241 CCATTCAGCC ATGACATTTC GACAGATTTG GTTGGTAGTG ATATCGCGAA AACCAAACAT
301 TTACCACCAA CGCTAATGGT GCAGTATTAT TTTGGCGAGT CTCAAAGTAA GTTCCGTCCA
361 TACGTTGGTG CAGGTCTGAA CTACACCATA TTCTTTGATG AAGATTTCAA TAGTACGGGT
421 AAAGGCGCTG ACCTGTCAGA TTTGAAATTA GATGATTCAT TCGGTCTAGC AGCGAATATT
481 GGTGTGGATT ACATGATCAA CGATCAATGG TTCCTCAACG CCGCTGCGTG GTACGCAAAT
541 ATTGAGACAG AAGCAACTTA CAAAGCAGGT GGAGTGAAGC AAAAAACCGA CGTCAAAATT
601 AACCCTTGGG TATTTATGAT CAGCGGCGGT TACAAGTTCT AA
The proteic aminoacid sequence of the said vibrio alginolyticus OmpW of the present invention is designated as SEQ ID NO.4, and its relevant information is:
(i) sequence signature:
(A) length: 213 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: polypeptide
(iii). sequence description:
1 MKKTICSLAV VAALVSPSVF AHKQGDFVLR VGAASVVPND SSDKILGSQE ELEVDSNTQL
61 GLTFGYMFTD NISLELLAAT PFSHDISTDL VGSDIAKTKH LPPTLMVQYY FGESQSKFRP
121 YVGAGLNYTI FFDEDFNSTG KGADLSDLKL DDSFGLAANI GVDYMINDQW FLNAAAWYAN
181 IETEATYKAG GVKQKTDVKI NPWVFMISGG YKF-
The preparation method of the said vibrio alginolyticus OmpW of the present invention gene, its step is as follows:
(1) nucleotide sequence that coding is had a purifying of vibrio alginolyticus OmpW protein active polypeptide operationally is connected in expression regulation sequence, forms molten alginic acid OmpW protein expression vector, and described nucleotide sequence is designated as the nucleotide sequence among the SEQ ID NO.3;
(2) change the expression vector in the step (1) over to host cell, form the proteic reconstitution cell of vibrio alginolyticus OmpW;
(3) be fit to express under the condition of vibrio alginolyticus OmpW protein polypeptide the reconstitution cell in the culturing step (2);
(4) isolate pure substantially polypeptide with vibrio alginolyticus OmpW protein-active.
The present invention and vibrio alginolyticus OmpW protein polypeptide specificity bonded antibody are polyclonal antibody.
Said " isolating ", " purifying " DNA are meant, this DNA or fragment are separated from the sequence that is arranged in its both sides under native state, also refer to this DNA or fragment with native state under the component of the nucleic acid followed separate, and separate with the protein of in cell, following it.
Said in the present invention carrier can be selected various carrier known in the art for use, and the carrier as commercially available comprises plasmid, clay etc.When producing vibrio alginolyticus OmpW polypeptide of the present invention, vibrio alginolyticus OmpW encoding sequence operationally can be connected in expression regulation sequence, thereby form vibrio alginolyticus OmpW protein expression vector.
Said " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjacent, then means in reading frame adjacent for the secretion leader sequence.
Said " host cell " is prokaryotic cell prokaryocyte.Prokaryotic host cell commonly used is intestinal bacteria (E.coli) or subtilis etc., preferred E.coli DH5 α; E.coli BL21 and E.coli TOP10 etc.
Can also analyze the expression of vibrio alginolyticus OmpW gene product with the Western engram analysis of vibrio alginolyticus OmpW specific antibody, and confirm its expression in biological specimen.
In addition, according to vibrio alginolyticus OmpW nucleotide sequence of the present invention and aminoacid sequence, can be on the homology basis of nucleic acid homology or marking protein, screening vibrio alginolyticus OmpW homologous gene or homologous protein.
In addition, proteic salt tolerant function of vibrio alginolyticus OmpW according to the present invention and molecular weight can screen vibrio alginolyticus OmpW homologous gene or homologous protein under different salinity.
Vibrio alginolyticus OmpW Nucleotide full length sequence of the present invention or its fragment can use pcr amplification method, recombination method or library screening method to obtain usually.For the pcr amplification method, can be disclosed according to the present invention relevant nucleotide sequence design primer, and with vibrio alginolyticus as template, amplification and must be about sequence.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available artificial chemosynthesis is synthesized relevant sequence.Before the application, prior art fully can be by first synthetic a plurality of polynucleotide small segments, and then connect and obtain code book and invent the proteic nucleotide sequence of molten alginic acid OmpW.Then, can be with in various existing dna moleculars (as carrier) and the cell in this nucleotide sequence introducing this area.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
Description of drawings
Fig. 1 is that vibrio alginolyticus outer membrane protein SDS-PAGE collection of illustrative plates compares under the different salinity condition.In Fig. 1,1-4 represent that respectively salinity is 1%, 2%, 3%, 4%, and M represents mark, and KDa represents the size of molecular weight.
Fig. 2 is under different salinity condition (0.85% and 3.5%), and vibrio parahaemolyticus outer membrane protein two-dimensional electrophoresis collection of illustrative plates relatively.
Fig. 3 is the abduction delivering of PET-OmpW clone in E.coli BL21.In Fig. 3,1 is the band of expression of institute's cloned genes; 2 is the band of empty carrier.
Fig. 4 identifies for the double digestion of PLLP-OmpA-OmpW clone.In Fig. 4,1-3 expression contrasts respectively, goal gene is two to be cut, mark, unit is Kb.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, the condition described in the Sambrook equimolecular cloning experimentation chamber handbook (2001 by Cold Spring Harbor Laboratory Press) for example, or the condition of advising according to manufacturer.
The clone of embodiment 1 vibrio alginolyticus OmpW gene
1. microbial culture
Vibrio alginolyticus derives from this laboratory and preserves bacterial strain, and picking list bacterium colony adds one 70 ℃ of preservations of 30% glycerine after the LB culture medium culturing is spent the night.
2. the full-length clone of gene (CIoning of full-length DNA)
From being that searching object can successfully be identified vibrio alginolyticus OmpW with vibrio parahaemolyticus gene group data, but from result's the not high this point of significance, between these two kinds of bacterium, still very high but OmpW should be variant similarity on sequence.Thus; can hold and add restriction enzyme site and two protection bases design primers according to the 5 ｀ end and 3 ｀ of Vibrio parahaemolyticus OmpW sequence; R1:5 ｀-GGGGATCCATGAAAAAAACAATCTG-3 ｀ (being designated as SEQ ID NO.1) is a forward primer; oligonucleotide R2:5 '-CCGAATTCTTAGAACTTGTAACCGC-3 ｀ (being designated as SEQ ID NO.2) is a reverse primer; with the vibrio alginolyticus is template; vibrio alginolyticus OmpW albumen is carried out pcr amplification; the PCR condition of R1/R2 be 94 ℃ 5 minutes; thereupon with 94 ℃ 1 minute; 55 ℃ of 1 minute and 72 ℃ carried out 35 circulations in 90 seconds, extended 5 minutes with 72 ℃ at last.The electrophoresis detection pcr amplification product, the acquisition expanding fragment length is 642bp.Clone, check order with pcr amplification product according to a conventional method then, obtain the sequence shown in the SEQ ID NO.3.
Above-mentioned SEQ ID NO.1 and SEQ ID NO.2 are respectively:
The information of SEQ ID NO.1:
(i) sequence signature:
(A) length: 25bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: oligonucleotide
(iii). sequence description: GGGGATCCATGAAAAAAACAATCTG
The information of SEQ ID NO.2:
(i) sequence signature:
(A) length: 25bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: oligonucleotide
(iii). sequence description: CCGAATTCTTAGAACTTGTAACCGC
The sequence information and the homology analysis of embodiment 2 vibrio alginolyticus OmpW genes
The new vibrio alginolyticus OmpW full length gene of the present invention is 642bp, and detailed sequence is seen SEQ ID NO.3.Derive the aminoacid sequence of vibrio alginolyticus OmpW according to the DNA total length, totally 213 amino-acid residues, molecular weight 22Kd, detailed sequence is designated as SEQ ID NO.4, and its relevant information is:
(i) sequence signature:
(A) length: 213 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: polypeptide
(iii). sequence description:
1 MKKTICSLAV VAALVSPSVF AHKQGDFVLR VGAASVVPND SSDKILGSQE ELEVDSNTQL
61 GLTFGYMFTD NISLELLAAT PFSHDISTDL VGSDIAKTKH LPPTLMVQYY FGESQSKFRP
121 YVGAGLNYTI FFDEDFNSTG KGADLSDLKL DDSFGLAANI GVDYMINDQW FLNAAAWYAN
181 IETEATYKAG GVKQKTDVKI NPWVFMISGG YKF-
Gene order total length and the based encode protein of OmpW are carried out Nucleotide and protein homology retrieval with blast program in ncbi database, found that there are very high homology in it and Vibrio parahaemolyticus OmpW gene.On nucleotide level, the 11-645 sequence in the whole coding sequence (GenBank Accession No.BA000032) of it and Vibrio parahaemolyticus OmpW gene has 88% homogeny (seeing Table 1):
Table 1
Percent Identity：88％in nt overlap
Query：11 caatctgcagtctagcagtggttgctgcactcgtgtcaccaagtgttttcgctcataaac 70
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：11 caatctgcagtctagcagtggttgctgcactcgtgtcaccaagtgttttcgctcataaac 70
Query：71 agggtgacttcgttcttcgtgttggtgcggcgtctgtcgttccaaatgacagcagtgata 130
| ||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||
Sbjct：71 aaggtgacttcgttcttcgtgttggtgcggcgtctgtcgttccaaatgacagcagcgata 130
Query：131 agattcttggttctcaagaagagttagaagttgactcaaatacgcagcttggtttgacgt 190
|||||||||||||||||||||| || |||| || |||||||||||||||||||||||||
Sbjct：131 agattcttggttctcaagaagaattgaaagtggattcaaatacgcagcttggtttgacgt 190
Query：191 ttggctacatgttcacagacaacatcagtttagagcttctagcagcaacaccattcagcc 250
|||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||
Sbjct：191 ttggctacatgttcacagacaacatcagcctagagcttctagcagcaacaccattcagcc 250
Query：251 atgacatttcgacagatttggttgg---tagtgatatcgcgaaaaccaaacatttaccac 307
|||| ||||| ||||| ||| | || | ||||||||||| | |||||||| | ||||
Sbjct：251 atgatatttcaacagacttgttaggtcttggtgatatcgcggacaccaaacaccttccac 310
Query：308 caacgctaatggtgcagtattattttggcgagtctcaaagtaagttccgtccatacgttg 367
||||||| ||||| ||||| || ||||||||| | |||||||||||||||||||||||||
Sbjct：311 caacgcttatggttcagtactactttggcgagccacaaagtaagttccgtccatacgttg 370
Query：368 gtgcaggtctgaactacaccatattctttgatgaagatttcaatagtacgggtaaaggcg 427
|||||||||| ||||||||||| || |||||||||| || || | | | ||| ||
Sbjct：371 gtgcaggtctcaactacaccatcttttttgatgaaggctttaacaacaaagcgaaaaacg 430
Query：428 ctgacctgtcagatttgaaattagatgattcattcggtctagcagcgaatattggtgtgg 487
| | | | ||| | || |||| |||||||| ||| ||||||| || | || ||||
Sbjct：431 tgggcttaactgatcttaagctagacgattcatttggtttagcagcaaacgtaggcgtgg 490
Query：488 attacatgatcaacgatcaatggttcctcaacgccgctgcgtggtacgcaaatattgaga547
| |||||||||||||||||||||||||| ||||| |||||||||| ||||| ||||| |
Sbjct：491 actacatgatcaacgatcaatggttccttaacgcatctgcgtggtatgcaaacattgaaa550
Query：548 cagaagcaacttacaaagcaggtggagtgaagcaaaaaaccgacgtcaaaattaaccctt607
|||||||||| |||||| ||||||| ||||||||||||||||||||||||||||||||
Sbjct：551 cagaagcaacatacaaatttggtggagcgaagcaaaaaaccgacgtcaaaattaaccctt610
Query：608 gggtatttatgatcagcggcggttacaagttctaa642
|||||||||||||||||||||||||||||||||||
Sbjct：611 gggtatttatgatcagcggcggttacaagttctaa645
Above-listed: the proteic nucleotide sequence of molten alginic acid OmpW
Following: the proteic nucleotide sequence of Vibrio parahaemolyticus OmpW (GenBank Accession No.BA000032)
On amino acid levels, the amino-acid residue of it and Vibrio parahaemolyticus OmpW albumen (GenPept Accession No.BAC61439) has high homogeny and similarity (seeing Table 2).Table 2 is that the homology of molten alginic acid OmpW albumen of the present invention and the proteic aminoacid sequence of Vibrio parahaemolyticus OmpW (GenPept Accession No.BAC61439) compares (FASTA) figure.Wherein, identical amino acid marks with the amino acid monocase between two sequences, and similar amino acid marks with "+".
Table 2
Identities＝181/214(84％)，Positives＝187/214(86％)，Gaps＝1/214(0％)
Query：1 MKKTICXXXXXXXXXXXXXXXHKQGDFVLRVGAASVVPNDSSDKILGSQEELEVDSNTQL 60
MKKTIC HKQGDFVLRVGAASVVPNDSSDKILGSQEEL+VDSNTQL
Sbjct：1 MKKTICSLAVVAALVSPSVFAHKQGDFVLRVGAASVVPNDSSDKILGSQEELKVDSNTQL 60
Query：61 GLTFGYMFTDNISLELLAATPFSHDISTDLVG-SDIAKTKHLPPTLMVQYYFGESQSKFR 119
GLTFGYMFTDNISLELLAATPFSHDISTDL+G DIA TKHLPPTLMVQYYFGE QSKFR
Sbjct：61 GLTFGYMFTDNISLELLAATPFSHDISTDLLGLGDIADTKHLPPTLMVQYYFGEPQSKFR 120
Query：120 PYVGAGLNYTIFFDEDFNSTGKGADLSDLKLDDSFGLAANIGVDYMINDQWFLNAAAWYA 179
PYVGAGLNYTIFFDE FN+ K L+DLKLDDSFGLAAN+GVDYMINDQWFLNA+AWYA
Sbjct：121 PYVGAGLNYTIFFDEGFNNKAKNVGLTDLKLDDSFGLAANVGVDYMINDQWFLNASAWYA 180
Query：180 NIETEATYKAGGVKQKTDVKINPWVFMISGGYKF 213
NIETEATYK GG KQKTDVKINPWVFMISGGYKF
Sbjct：181 NIETEATYKFGGAKQKTDVKINPWVFMISGGYKF 214
Above-listed: the proteic aminoacid sequence of molten alginic acid OmpW
Following: the proteic aminoacid sequence of Vibrio parahaemolyticus OmpW (GenPept Accession No.BAC61439)
Therefore all there are higher homology in vibrio alginolyticus OmpW gene and Vibrio parahaemolyticus OmpW gene on nucleic acid still is protein level, can think that both also have very high similarity on function.
The proteic 26S Proteasome Structure and Function research of embodiment 3 vibrio alginolyticus OmpW
Below provide the variation of vibrio alginolyticus OmpW under the different salt concentration conditions:
Having extracted vibrio alginolyticus after the outer membrane protein under 1%, 2%, 3% and 4% these four kinds of salt concn culture condition, at first their outer membrane protein collection of illustrative plates are compared analysis by SDS-PAGE, the result is as shown in Figure 1.From Fig. 1, can be clear that, along with the rising gradually of salinity, have a protein band also respective table reveal the amount on rising gradually, this salt adaptability that shows this protein band and vibrio alginolyticus has the property of being closely related.Can know that in conjunction with qualification result this band is OmpW to vibrio alginolyticus outer membrane protein.
Below provide the variation of Vibrio parahaemolyticus OmpW under the different salt concentration conditions:
Similar with research to vibrio alginolyticus, the expression of Vibrio parahaemolyticus outer membrane protein under the different salinity condition is compared analysis.Extract the outer membrane protein of the Vibrio parahaemolyticus that (0.85% and 3.5%) is cultivated under the different salinity condition, they have been carried out the two-dimensional electrophoresis atlas analysis, the result as shown in Figure 2.
In analysis to two kinds of salinity outer membrane protein differential expressions, can find a differential expression point (marking) as Fig. 2, its qualification result also is OmpW, is consistent with situation in the vibrio alginolyticus.As can be seen from Figure 2, OmpW is along with the rising expression amount of salinity also improves, and this also is consistent with situation in the vibrio alginolyticus.This is further illustrated in the dependency between the OmpW albumen and salt adaptive faculty in the vibrios.
Simultaneously from Fig. 2 also as can be seen, the expression amount of OmpW under the high salinity situation is far below the expression amount at vibrio alginolyticus in the Vibrio parahaemolyticus, if two kinds of albumen and salt adaptability are closely related, may have influence on the difference of bacterium in its expression amount difference so to the salinity tolerance.
Below provide of the comparison of three kinds of vibrios to salt tolerance level ability:
Except above vibrio alginolyticus and Vibrio parahaemolyticus, can add a bacterial classification-Vibrio vulnificus highly significant and together analyze.Why saying that Vibrio vulnificus is meaningful, is because of after the genome of Vibrio vulnificus is analyzed, and finds that there is not the OmpW gene in it, and it is carried out the salt tolerance relatively with preceding two kinds of bacterium, can be clear that the effect of OmpW more.
On the LB of 13% salt concn flat board, inserted these three kinds of vibrios simultaneously 1%, 2%, whether can grow according to bacterium on the different salinity flat board and judge that the difference of their salt tolerance levels, table 3 are the results that observe after 18 hours in inoculation.
Table 3
| Salinity (%) | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| V.vulnificus | + | + | + | - | - | - | - | - | - | - | - | - |
| V.parahaemolyticus | + | + | + | + | + | + | + | + | ||||
| V.alginolyticus | + | + | + | + | + | + | + | + | + | + | + | + |
Annotate :+expression has bacterium colony;-expression is not grown
Result from table 3 as can be seen, on the salt tolerance, vibrio alginolyticus〉Vibrio parahaemolyticus Vibrio vulnificus, this situation with whether express OmpW albumen and expression amount in these three kinds of vibrios different just in time corresponding mutually, this further shows the dependency of OmpW and vibrios salt tolerance.Below further prove the mutual relationship between OmpW albumen and salt tolerance.
Behind proteic clone of vibrio alginolyticus OmpW and the abduction delivering to the influence of thalline salt tolerance:
After by the method among the embodiment 1 vibrio alginolyticus OmpW albumen being carried out pcr amplification, be connected respectively among PETHIS and the PLLP-OmpA and finally change over to respectively among E.coli BL21 and the Top10.Fig. 3 is to the SDS-PAGE collection of illustrative plates of E.coli BL21 OmpW transformant behind the IPTG abduction delivering.From Fig. 3, can be clear that the proteic expression of OmpW.Through the bacterial strain of abduction delivering and carried out the analysis of salt tolerance through inductive E.coli Top10+PLLP-OmpA-OmpW equally, table 4 is in each the observation on Growth result of salinity plating after 24 hours to this.
Table 4
| Salinity (1%) | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| E.coli BL21+PET-OmpW | + | + | + | + | + | - | |
| E.coli BL21+PET-OmpW | + | + | + | + | + | - | |
| E.coli Top10+pLLP-OmpA | + | + | + | + | + | - | - |
| E.coli Top10+pLLP-OmpA-OmpW | + | + | + | + | + | + | + |
As can be seen from Table 4, E.coli Top10+PLLP-OmpA-Omp has compared according to height on the tolerance of strain E.coli Top10+PLLP-OmpA salt two salinity, and this has obviously shown the directly related property of vibrio alginolyticus OmpW with the salt tolerance.
Simultaneously, can see that E.coli BL21+PET-OmpW and its contrast strain E.coli BL21+PET-OmpW are basic identical on the salt tolerance level, not show OmpW and import the lifting of back its salt resistance ability.Its reason mainly is that OmpW still is present in the born of the same parents after expressing in E.coli BL21+PET-OmpW, is difficult to arrive the adventitia position, thereby can't brings into play its effect aspect the salt tolerance.And after using the PLLP-OmpA carrier, since on the carrier with the effect of OmpA signal peptide, the OmpW albumen of abduction delivering can be secreted into outside the born of the same parents, and OmpW albumen just shows it in the effect that promotes on the salt tolerance after inserting adventitia by certain mode.The Different Results of using these two kinds of carriers to draw further shown, OmpW expresses that to cause the lifting of salt tolerance level be the effect of OmpW albumen self, rather than causes other variation of cell behind its abduction delivering and produce.
The preparation and the purification of embodiment 4 vibrio alginolyticus OmpW polypeptide
The vibrio alginolyticus OmpW encoding sequence of total length or fragment are built among the Escherichia coli protein prokaryotic expression carrier, express and the purification target protein to reach.
The structure of bacillus coli DH 5 alpha cloning vector, and transform DH5 α competent cell:
According to the above primer that designs, correspond respectively to about 20 Nucleotide of encoding sequence 5 ' and 3 ' end, and on positive anti-primer, introduce restricted endoenzyme site (this decides according to the colibacillus expression plasmid PET-HIS that selects for use) respectively, so that make up clone and expression vector.With the amplified production that obtains among the embodiment 1 is template, behind pcr amplification, with vibrio alginolyticus OmpW gene clone to E.coli expression plasmid PET-HIS. with suitable restriction endonuclease with plasmid linearization.Determine the purity and the concentration of plasmid with agarose gel electrophoresis.With linearizing cloned plasmids, be transformed into cloning vector E.coli with chemical heat shock method
In the DH5 α competence.All be applied on the LB+Amp flat board, cultivated 12～16 hours for 37 ℃.
The clone advances screening, the evaluation of the proteic DH5 α of molten alginic acid bacterium OmpW positive colony:
Intestinal bacteria bacterium colony on the picking LB+Amp flat board at random, be inoculated on the LB+Amp liquid nutrient medium, inoculation contains simultaneously the DH5 of free PET-HIS plasmid α, incubated overnight, extract plasmid respectively with alkaline lysis, and the plasmid that is extracted is carried out double digestion respectively verify, obtain the double digestion collection of illustrative plates as shown in Figure 4.
The structure of e. coli bl21 expression vector, and transform the BL21 competent cell:
The above plasmid that contains vibrio alginolyticus OmpW protein gene that detects is transformed into BL21 competence with same chemical heat shock method.All be applied on the LB+Amp flat board, cultivated 10-16 hours for 37 ℃.
Express screening, the evaluation of the proteic BL21 positive colony of vibrio alginolyticus OMPW:
E. coli bl21 bacterium colony on the picking LB+Amp flat board at random, be inoculated on the LB+Amp liquid nutrient medium, inoculate unconverted BL21 and the BL21 that contains the PET-HIS empty plasmid simultaneously, in 30 ℃ of moderate jogs to OD value is 0.6, adding IPTG100g/ml35 ℃ induced two hours, centrifugal collection thalline, SDS-PAGE detects expression product, and the result is as shown in Figure 2.
Utilize Western blot to detect the expression of OmpW albumen in transgenosis E.coli:
1, proteic obtaining: express vibrio alginolyticus OmpW albumen as stated above.
2, the preparation of SDS-PAGE protein isolate: SDS-PAGE is with reference to " molecular cloning " (Sambrook etc., 2001); A) before the application of sample, sample is placed LSB sample loading buffer (2* sample loading buffer: glycerine 2.4g, 1M Tris-HCl pH6.81ml; Bromjophenol blue 0.01%, H2O is settled to 20ml), boiled 3～5 minutes; B) polyacrylamide gel electrophoresis under 4 ℃ of 80V voltage reaches the gel bottom up to indicator (bromjophenol blue) forward position.
3, protein is to shifting on the nitrocellulose membrane: before a) shifting, use transfering buffering liquid (39mmol glycine, 48mmolTris Base, 0.037% SDS, 20% methyl alcohol) balanced gel and nitrocellulose membrane 30 minutes; B) 4 ℃ down with DYY-7B electroporation 50V transferase 12 hour, and 3 layers of Whatman filter paper are respectively filled up in the gel both sides.
4, proteic detection on the film: a) nitrocellulose membrane is immersed in the confining liquid, 60 minutes (confining liquids: get the 5g skim-milk and be dissolved in 100ml TNT are slowly shaken, sealed to room temperature; B) again film is immersed in the lavation buffer solution room temperature washing three times, each 10 minutes; C) add first antibody (antibody of anti-vibrio alginolyticus OmpW), room temperature reaction 60 minutes; Same step b) is washed three times; D) add second antibody (anti-mouse two resists the horseradish peroxidase rabbit), room temperature reaction 60 minutes; Same step b) is washed three times; E) add substrate DAB colour developing and observe protein band.
Claims (12)
1, Vibrio alginolocius OmpW gene is characterized in that it being the nucleotide sequence that coding has the polypeptide of vibrio alginolyticus OmpW protein active, is designated as SEQ ID NO.3, promptly
(i) sequence signature:
(A) length: 642bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: Nucleotide
(iii). sequence description: SEQ ID NO.3
1 ATGAAAAAAA CAATCTGCAG TCTAGCAGTG GTTGCTGCAC TCGTGTCACC AAGTGTTTTC
61 GCTCATAAAC AGGGTGACTT CGTTCTTCGT GTTGGTGCGG CGTCTGTCGT TCCAAATGAC
121 AGCAGTGATA AGATTCTTGG TTCTCAAGAA GAGTTAGAAG TTGACTCAAA TACGCAGCTT
181 GGTTTGACGT TTGGCTACAT GTTCACAGAC AACATCAGTT TAGAGCTTCT AGCAGCAACA
241 CCATTCAGCC ATGACATTTC GACAGATTTG GTTGGTAGTG ATATCGCGAA AACCAAACAT
301 TTACCACCAA CGCTAATGGT GCAGTATTAT TTTGGCGAGT CTCAAAGTAA GTTCCGTCCA
361 TACGTTGGTG CAGGTCTGAA CTACACCATA TTCTTTGATG AAGATTTCAA TAGTACGGGT
421 AAAGGCGCTG ACCTGTCAGA TTTGAAATTA GATGATTCAT TCGGTCTAGC AGCGAATATT
481 GGTGTGGATT ACATGATCAA CGATCAATGG TTCCTCAACG CCGCTGCGTG GTACGCAAAT
541 ATTGAGACAG AAGCAACTTA CAAAGCAGGT GGAGTGAAGC AAAAAACCGA CGTCAAAATT
601 AACCCTTGGG TATTTATGAT CAGCGGCGGT TACAAGTTCT AA。
2, the proteic aminoacid sequence of vibrio alginolyticus OmpW is characterized in that being designated as SEQ ID NO.4, and its relevant information is:
(i) sequence signature:
(A) length: 213 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: polypeptide
(iii). sequence description:
1 MKKTICSLAV VAALVSPSVF AHKQGDFVLR VGAASVVPND SSDKILGSQE ELEVDSNTQL
61 GLTFGYMFTD NISLELLAAT PFSHDISTDL VGSDIAKTKH LPPTLMVQYY FGESQSKFRP
121 YVGAGLNYTI FFDEDFNSTG KGADLSDLKL DDSFGLAANI GVDYMINDQW FLNAAAWYAN
181 IETEATYKAG GVKQKTDVKI NPWVFMISGG YKF。
3, the preparation method of vibrio alginolyticus OmpW gene is characterized in that its step is as follows:
5` end and 3` according to Vibrio parahaemolyticus OmpW sequence hold and add restriction enzyme site and two protection bases design primers; R1:5`-GGGGATCCATGAAAAAAACAATCTG-3` is a forward primer; be designated as SEQ ID NO.1; oligonucleotide R2:5 '-CCGAATTCTTAGAACTTGTAACCGC-3` is a reverse primer; be designated as SEQ ID NO.2; with the vibrio alginolyticus is template; vibrio alginolyticus OmpW albumen is carried out pcr amplification; the PCR condition of R1/R2 be 94 ℃ 5 minutes; thereupon with 94 ℃ 1 minute; 55 ℃ of 1 minute and 72 ℃ carried out 35 circulations in 90 seconds; extended 5 minutes with 72 ℃ at last; the electrophoresis detection pcr amplification product; the acquisition expanding fragment length is 642bp, clones with pcr amplification product according to a conventional method then; order-checking obtains the sequence shown in the SEQ ID NO.3.
4, the preparation method who has the polypeptide of vibrio alginolyticus OmpW protein-active is characterized in that its step is as follows:
(1) nucleotide sequence that coding is had a purifying of vibrio alginolyticus OmpW protein active polypeptide operationally is connected in expression regulation sequence, forms molten alginic acid OmpW protein expression vector, and described nucleotide sequence is designated as the nucleotide sequence among the SEQ ID NO.3;
(2) change the expression vector in the step (1) over to host cell, form the proteic reconstitution cell of vibrio alginolyticus OmpW;
(3) be fit to express under the condition of vibrio alginolyticus OmpW protein polypeptide the reconstitution cell in the culturing step (2);
(4) isolate polypeptide with vibrio alginolyticus OmpW protein-active;
Described nucleotide sequence is designated as SEQ ID NO.3, promptly
(i) sequence signature:
(A) length: 642bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: Nucleotide
(iii). sequence description: SEQ ID NO.3
1 ATGAAAAAAA CAATCTGCAG TCTAGCAGTG GTTGCTGCAC TCGTGTCACC AAGTGTTTTC
61 GCTCATAAAC AGGGTGACTT CGTTCTTCGT GTTGGTGCGG CGTCTGTCGT TCCAAATGAC
121 AGCAGTGATA AGATTCTTGG TTCTCAAGAA GAGTTAGAAG TTGACTCAAA TACGCAGCTT
181 GGTTTGACGT TTGGCTACAT GTTCACAGAC AACATCAGTT TAGAGCTTCT AGCAGCAACA
241 CCATTCAGCC ATGACATTTC GACAGATTTG GTTGGTAGTG ATATCGCGAA AACCAAACAT
301 TTACCACCAA CGCTAATGGT GCAGTATTAT TTTGGCGAGT CTCAAAGTAA GTTCCGTCCA
361 TACGTTGGTG CAGGTCTGAA CTACACCATA TTCTTTGATG AAGATTTCAA TAGTACGGGT
421 AAAGGCGCTG ACCTGTCAGA TTTGAAATTA GATGATTCAT TCGGTCTAGC AGCGAATATT
481 GGTGTGGATT ACATGATCAA CGATCAATGG TTCCTCAACG CCGCTGCGTG GTACGCAAAT
541 ATTGAGACAG AAGCAACTTA CAAAGCAGGT GGAGTGAAGC AAAAAACCGA CGTCAAAATT
601 AACCCTTGGG TATTTATGAT CAGCGGCGGT TACA AGTTCT AA。
5, the preparation method with polypeptide of vibrio alginolyticus OmpW protein-active as claimed in claim 4 is characterized in that described carrier is plasmid or cosmid vector.
6, the preparation method with polypeptide of vibrio alginolyticus OmpW protein-active as claimed in claim 4 is characterized in that in step 2) in, described host cell is a prokaryotic cell prokaryocyte.
7, the preparation method with polypeptide of vibrio alginolyticus OmpW protein-active as claimed in claim 6 is characterized in that described prokaryotic cell prokaryocyte is intestinal bacteria or subtilis.
8, the preparation method with polypeptide of vibrio alginolyticus OmpW protein-active as claimed in claim 7 is characterized in that described intestinal bacteria are selected from bacillus coli DH 5 α, e. coli bl21 or intestinal bacteria TOP10.
9, the application of Vibrio alginolocius OmpW gene as claimed in claim 1 in preparation salt tolerant product.
10, the application of vibrio alginolyticus OmpW albumen as claimed in claim 2 in preparation salt tolerant product.
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| CN103031358B (en) * | 2012-08-06 | 2015-07-22 | 浙江理工大学 | Method for screening pharmaceuticals through taking escherichia coli outer membrane protein gene OmpW as target spot |
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| Proteomic analysis of salt-sensitive outer membrane proteinsof Vibrio parahaemolyticus. Xu, CX, Ren, HX, Wang, SY, et al.RES MICROBIOL,Vol.155 No.10. 2004 * |
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