CN100344649C - Mermaid luminous bacillus outer membrane protein V and coding sequence, its preparation method and application - Google Patents
Mermaid luminous bacillus outer membrane protein V and coding sequence, its preparation method and application Download PDFInfo
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- CN100344649C CN100344649C CNB2005101002132A CN200510100213A CN100344649C CN 100344649 C CN100344649 C CN 100344649C CN B2005101002132 A CNB2005101002132 A CN B2005101002132A CN 200510100213 A CN200510100213 A CN 200510100213A CN 100344649 C CN100344649 C CN 100344649C
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- luminous bacillus
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- mermaid luminous
- membrane protein
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Abstract
The present invention provides novel mermaid photobacteria outer membrane protein V, a nucleotide sequence and an amino acid sequence of the mermaid photobacteria outer membrane protein V for encoding a polypeptide with the protein activity of the mermaid photobacteria outer membrane protein V, and a method for preparing the mermaid photobacteria outer membrane protein V by recombinant technology. The present invention also provides the application of the mermaid photobacteria OmpV protein and an encoding sequence thereof.
Description
Technical field
The invention belongs to biological technical field, especially relate to a kind of new coding Mermaid luminous bacillus (Photobacteriumdamsela) outer membrane protein V (outer membrane protein V, OmpV) polynucleotide, and the polypeptide of this polynucleotide encoding, also relate to the preparation method and its usage of these polynucleotide and polypeptide.
Background technology
Bacillus is to study maximum a kind of gram negative bacteriums at present, and gram negative bacterium has very strong adaptability and resistance because of its unique cell wall structure.The gram negative bacterium great majority are conditioned pathogen, can cause multiple infection when immunity of organisms is low, have a lot of diseases to be caused by gram negative bacterium in the mankind and the livestock.Main pathogenic bacillus has dysentery bacterium, Corynebacterium diphtheriae, intestinal bacteria, Bacillus proteus, Pseudomonas aeruginosa, bordetella pertussis etc., and they produce intracellular toxin, by intracellular toxin the people is caused a disease.1996 in Japan the maximum in the world eruption and prevalence that is caused by O157:H7 intestinal bacteria have taken place once.(Escherichia coli) is very wide in distributed in nature for intestinal bacteria, is the normal microflora in the humans and animals enteron aisle.Generally not pathogenic under the normal circumstances, but it is a conditioned pathogen.In recent years, worldwide infectation of bacteria type and bacterial infection have considerable change, originally threaten maximum germ to be replaced by nonpathogenic weak toadstool and conditioned pathogen just gradually to the human life.Mermaid luminous bacillus is exactly this bacterioid of relatively paying attention at present.Mermaid luminous bacillus is a kind of Gram-negative, can be luminous, facultative Bacteroides nodosus, it is 1981 at first, from the ulcer wound of the catfish that warm sea water, grows, be separated to, and be categorized into Vibrio, be called the mermaid vibrios, because people and animals' disease that it can cause is similar to Vibrio, all can cause human gastroenteritis, wound infection and people and animals' septicemia (CURRENT MICROBIOLOGY Vol.48 (2004), pp.167-174 Cloning and Characterization of the Gene Encoding for OMP-PDPorin:The Major Photobacterium damsela Outer Membrane Protein).At comparison and chromosomal DNA-DNA hybridization data based on the 16sRNA gene order in 1991 it is referred to Bacillaceae, be called Mermaid luminous bacillus (Shin JH, Shin MG, Suh SP, Ryang DW, Rew JS, Nolte FS.Primary Vibrio damselasepticemia.Clin Infect Dis 1996; 22:856-857.).It is a kind of bacterium that causes the tissue necrosis transmissible disease of tool extreme damage, and it not only can cause pseudotuberculosis and fish hueppe's disease, causes aquatic products to culture loss more than 50%; Can also the people be caused a disease by people's wound or edible underdone sea-food, infect Mermaid luminous bacillus and serious morbific incident with regard to a lot of fishermen are taken place in Japan.More fearful is, increasing report shows that Mermaid luminous bacillus has strong lethality, can be simultaneously so that immune deficiency or healthy factitious host to be arranged, and bring out fast, fulminant infection (Fraser SL, Purcell BK, Delgado B Jr, Baker AE, Whelen AC.Rapidly fatal infectiondue to Photobacterium (Vibrio) damsela.Clin Infect Dis 1997; 25:935-936.).
OmpV is an important albumen in the bacterial outer membrane albumen, and proteic research is considerably less to OmpV at present.In all bacteriums, only obtain the gene order of 3 kinds of bacteriums such as Salmonella enterica, vibrio cholerae and Vibrio parahaemolyticus.For its 26S Proteasome Structure and Function, only think just that OmpV may be a porin, be one can be thermoinducible, so the albumen OmpV of tool high immunogenicity is an albumen that merits attention very much and further investigate.
Shang Weijian has any Mermaid luminous bacillus OmpV nucleotide sequence of mentioning that discloses or reported.Do not see yet and reported that bacillus OmpV albumen had the salt sensitive function.
Summary of the invention
The object of the present invention is to provide a kind of new Mermaid luminous bacillus outer membrane protein V.
Another object of the present invention provides the nucleotide sequence that a kind of coding has the polypeptide of Mermaid luminous bacillus outer membrane protein V protein active.
The 3rd purpose of the present invention provides a kind of method of utilizing recombinant technology to prepare above-mentioned new Mermaid luminous bacillus outer membrane protein V.
The present invention also provides the application of this Mermaid luminous bacillus outer membrane protein V and encoding sequence, and this proteic a kind of new function promptly is provided, i.e. salt sensitive function is in the application that improves the biomass cells brine sensitivity.
The said Mermaid luminous bacillus outer membrane protein V of the present invention is characterized in that: this polypeptide contains the aminoacid sequence of SEQ ID NO.4.
The aminoacid sequence of above-mentioned Mermaid luminous bacillus outer membrane protein V is shown in SEQ ID NO.4, and its relevant information is:
(i) sequence signature:
(A) length: 258 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: polypeptide
(iii). sequence description:
MNKTLIALLT LAAAGTATAG DTYIRNGNIY NNQGGWVAEV GVAQGSDLFK DQKHNTAPIL 60
NGGYHGEDFN ADLGGTNYRF LGETNDLINM SAYVVGSGII RDGDVAKSLK GTQKRRLAVD 120
LGLNTDFTLD EHNVISTYLQ HDISGAYKGY LAGATYFHIM NFGSVDFVPF ANLTYQSEDY 180
VDYYFGIKDR EATANRKAYK GDATVSYGLG YKLVMPINDN WQINQTTQYT RLGSGISDSS 240
VVDSANOWVV GASVSYNF 258
The said isolating polynucleotide of the present invention, it comprises a nucleotide sequence, and this nucleotide sequence is selected from down group:
(a) coding contains the polynucleotide of polypeptide of the aminoacid sequence of SEQ ID NO.4;
(b) with polynucleotide (a) complementary polynucleotide.
Above-mentioned polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO.4.
The sequence of above-mentioned polynucleotide is selected from coding region sequence or the full length sequence of SEQ ID NO.3, and its relevant information is:
(i) sequence signature:
(A) length: 777bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: Nucleotide
(iii). sequence description: SEQ ID NO.3
atgaacaaga cacttatcgc actactaact ttggcagcag ctggcaccgc aacggcaggt 60
gacacataca tccgtaacgg caacatctac aacaaccaag gtggttgggt tgcggaagtg 120
ggcgtagctc aaggtagtga cctatttaaa gatcaaaaac acaatactgc accaatccta 180
aacggtggtt accacggtga agacttcaac gcagatttgg gcggcaccaa ctaccgcttc 240
ttgggtgaaa ccaacgatct gatcaacatg agcgcatacg tggtaggcag cggcattatt 300
cgtgatggcg atgtagcaaa gagcttgaaa ggcacacaaa aacgtcgttt agcggttgat 360
ttaggtctaa ataccgattt cactctagat gagcacaacg tcatctcgac atacctacaa 420
cacgacatca gcggcgcata caaaggttac ttagcgggcg cgacgtactt ccacatcatg 480
aacttcggta gtgttgattt tgttcctttt gctaacctaa cttatcaaag tgaagactac 540
gtagattact actttggcat caaagatcgt gaagcgacgg caaaccgcaa agcatacaaa 600
ggtgatgcaa cggtaagcta cggtctgggc tacaagctgg ttatgccaat taatgacaac 660
tggcaaatca accaaacaac tcaatacact cgcctaggca gtggtatcag tgattcatct 720
gttgtagata gcgcaaacca atgggttgtt ggcgcaagcg tgtcttacaa cttctaa 777
The preparation method of the said Mermaid luminous bacillus outer membrane protein V of the present invention, its step is as follows:
(1) nucleotide sequence that coding is had a Mermaid luminous bacillus OmpV protein active polypeptide operationally is connected in expression regulation sequence, forms Mermaid luminous bacillus OmpV protein expression vector;
(2) change the expression vector in the step (1) over to host cell, form the proteic reconstitution cell of Mermaid luminous bacillus OmpV;
(3) be fit to express under the condition of Mermaid luminous bacillus OmpV protein polypeptide, the reconstitution cell in the culturing step (2) is expressed target product;
(4) separation and purification has the polypeptide of Mermaid luminous bacillus OmpV protein-active.
The present invention and Mermaid luminous bacillus OmpV 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 Mermaid luminous bacillus OmpV polypeptide of the present invention, Mermaid luminous bacillus OmpV encoding sequence operationally can be connected in expression regulation sequence, thereby form Mermaid luminous bacillus OmpV 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 Mermaid luminous bacillus OmpV gene product with the Western engram analysis of Mermaid luminous bacillus OmpV specific antibody, and confirm its expression in biological specimen.
In addition, according to Mermaid luminous bacillus OmpV nucleotide sequence of the present invention and aminoacid sequence, can be on the homology basis of nucleic acid homology or marking protein, screening Mermaid luminous bacillus OmpV homologous gene or homologous protein.
In addition, proteic salt sensitive function of Mermaid luminous bacillus OmpV according to the present invention and molecular weight can screen Mermaid luminous bacillus OmpV homologous gene or homologous protein under different salinity.
Mermaid luminous bacillus OmpV 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 Mermaid luminous bacillus 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 the proteic nucleotide sequence of code book invention Mermaid luminous bacillus OmpV.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 Mermaid luminous bacillus outer membrane protein SDS-PAGE collection of illustrative plates compares under the different salinity condition.In Fig. 1,1-4 represents that respectively salinity is 0.5%, 1%, 4%, and M represents mark, and KDa represents the size of molecular weight.
Fig. 2 is under different salinity condition (0.5%, 1%, 4%), and Mermaid luminous bacillus outer membrane protein two-dimensional electrophoresis collection of illustrative plates relatively.
Fig. 3 is the abduction delivering of PET-OmpV clone in E.coli BL21.In Fig. 3,2,3 is the band of expression of institute's cloned genes; 1 is the band of empty carrier.
Fig. 4 identifies for the PCR of PLLP-OmpA-OmpW clone.In Fig. 4,1 negative contrast, 2 positive contrasts, 3～10 positive clones' PCR identifies that 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 (2001by Cold Spring Harbor Laboratory Press) for example, or the condition of advising according to manufacturer.
The clone of embodiment 1 Mermaid luminous bacillus OmpV gene
1. acquisition of bacterium and cultivation
The various bacteriums that obtain from the marine site after simple separation and purification, obtain one with vibrios special culture media TCBS preliminary screening and are green bacterium colony, are decided to be Mermaid luminous bacillus after the VITEK32 of Xiamen City Disease Control and Prevention Center type automatic bacterial assessing instrument is identified.
Picking Mermaid luminous bacillus list bacterium colony adds 30% glycerine-70 ℃ preservation after the LB culture medium culturing is spent the night.
2. the full-length clone of gene (Cloning of full-length DNA)
Because the complete sequence of Mermaid luminous bacillus gene is undetermined also, there is not the gene order report of this bacterium OmpV at present yet.In view of Vibrio parahaemolyticus has been finished complete sequence determination, we by its OmpV sequences Design OmpV gene primer, be used for the pcr amplification of Mermaid luminous bacillus OmpV.Thus, can hold and add restriction enzyme site and two protection bases design primers, R1:5 '-GG according to the 5` end and the 3` of Vibrio parahaemolyticus OmpV sequence
GGATCCATGAACAAGACACT-3 ' (being designated as SEQ ID NO.1) is a forward primer, oligonucleotide R2:5 '-CC
GCTAGCTTAGAAGTTGTAAGACAC-3 ' (being designated as SEQ ID NO.2) is a reverse primer, with the Mermaid luminous bacillus is template, Mermaid luminous bacillus OmpV albumen is carried out pcr amplification, the PCR condition of R1/R2 be 94 ℃ 5 minutes, carried out 35 circulations in 90 seconds with 94 ℃ 1 minute, 55 ℃ 1 minute and 72 ℃ thereupon, extended 5 minutes with 72 ℃ at last.The electrophoresis detection pcr amplification product, the acquisition expanding fragment length is 777bp.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: 22bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: oligonucleotide
(iii). sequence description: GG
GGATCCATGAACAAGACACT
The information of SEQ ID NO.2:
(i) sequence signature:
(A) length: 26bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: oligonucleotide
(iii). sequence description: CC
GCTAGCTTAGAAGTTGTAAGACAC
The sequence information and the homology analysis of embodiment 2 Mermaid luminous bacillus OmpV genes
The new Mermaid luminous bacillus OmpV full length gene of the present invention is 777bp, and detailed sequence is seen SEQ ID NO.3.Derive the aminoacid sequence of Mermaid luminous bacillus OmpV according to the DNA total length, totally 258 amino-acid residues, molecular weight 27kD, detailed sequence is designated as SEQ ID NO.4, and its relevant information is:
(i) sequence signature:
(A) length: 258 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: polypeptide
(iii). sequence description:
MNKTLIALLT LAAAGTATAG DTYIRNGNIY NNQGGWVAEV GVAQGSDLFK DQKHNTAPIL 60
NGGYHGEDFN ADLGGTNYRF LGETNDLINM SAYVVGSGII RDGDVAKSLK GTQKRRLAVD 120
LGLNTDFTLD EHNVISTYLQ HDISGAYKGY LAGATYFHIM NFGSVDFVPF ANLTYQSEDY 180
VDYYFGIKDR EATANRKAYK GDATVSYGLG YKLVMPINDN WQINQTTQYT RLGSGISDSS 240
VVDSANQWVV GASVSYNF 258
Gene order total length and the based encode protein of OmpV 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 OmpV gene.On nucleotide level, the 11-645 sequence in the whole coding sequence (GenBank Accession No.BA000032) of it and Vibrio parahaemolyticus OmpV gene has 99% homogeny (seeing Table 1):
Table 1
Percent Identity：99％in nt overlap
Query：1 atgaacaagacacttatcgcactactaactttggcagcagctggcaccgcaacggcaggt 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：1 atgaacaagacacttatcgcactactaactttggcagcagctggcaccgcaacggcaggt 60
Query：61 gacacatacatccgtaacggcaacatctacaacaaccaaggtggttgggttgcggaagtg 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：61 gacacatacatccgtaacggcaacatctacaacaaccaaggtggttgggttgcggaagtg 120
Query：121 ggcgtagctcaaggtagtgacctatttaaagatcaaaaacacaatactgcaccaatccta 180
|||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||
Sbjct：121 ggcgtagctcaaggtagtgacctatttaaagatcaaaaacacaatactgcaccaattcta 180
Query：181 aacggtggttaccacggtgaagacttcaacgcagatttgggcggcaccaactaccgcttc 240
|||||||||||||||||||||||||||||||||||||||||||||| |||||||||||||
Sbjct：181 aacggtggttaccacggtgaagacttcaacgcagatttgggcggcatcaactaccgcttc 240
Query：241 ttgggtgaaaccaacgatctgatcaacatgagcgcatacgtggtaggcagcggcattatt 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：241 ttgggtgaaaccaacgatctgatcaacatgagcgcatacgtggtaggcagcggcattatt 300
Query：301 cgtgatggcgatgtagcaaagagcttgaaaggcacacaaaaacgtcgtttagcggttgat 360
||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||
Sbjct：301 cgtgatggcgatgtagcaaagagcttgaaaggcacacaaaaacgtcgtttagcagttgat 360
Query：361 ttaggtctaaataccgatttcactctagatgagcacaacgtcatctcgacatacctacaa 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：361 ttaggtctaaataccgatttcactctagatgagcacaacgtcatctcgacatacctacaa 420
Query：421 cacgacatcagcggcgcatacaaaggttacttagcgggcgcgacgtacttccacatcatg 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：421 cacgacatcagcggcgcatacaaaggttacttagcgggcgcgacgtacttccacatcatg 480
Query：481 aacttcggtagtgttgattttgttccttttgctaacctaacttatcaaagtgaagactac 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：481 aacttcggtagtgttgattttgttccttttgctaacctaacttatcaaagtgaagactac 540
Query：541 gtagattactactttggcatcaaagatcgtgaagcgacggcaaaccgcaaagcatacaaa 600
|||||||||||||||||||||||||| |||||||||||||||||||||||||||||||||
Sbjct：541 gtagattactactttggcatcaaagaccgtgaagcgacggcaaaccgcaaagcatacaaa 600
Query：601 ggtgatgcaacggtaagctacggtctgggctacaagctggttatgccaattaatgacaac 660
||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：601 ggtgatgcaactgtaagctacggtctgggctacaagctggttatgccaattaatgacaac 660
Query：661 tggcaaatcaaccaaacaactcaatacactcgcctaggcagtggtatcagtgattcatct 720
||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||
Sbjct：661 tggcaaatcaaccaaacgactcaatacactcgcctaggcagtggtatcagtgattcatct 720
Query：721 gttgtagatagcgcaaaccaatgggttgttggcgcaagcgtgtcttacaacttctaa 777
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct：721 gttgtagatagcgcaaaccaatgggttgttggcgcaagcgtgtcttacaacttctaa 777
Above-listed: the proteic nucleotide sequence of Mermaid luminous bacillus OmpV
Following: the proteic nucleotide sequence of Vibrio parahaemolyticus OmpV (GenBank Accession No.BA000032)
On amino acid levels, the amino-acid residue of it and Vibrio parahaemolyticus OmpV albumen (GenPept Accession No.BAC61661) has high homogeny and similarity (seeing Table 2).Table 2 is that the homology of Mermaid luminous bacillus OmpV albumen of the present invention and the proteic aminoacid sequence of Vibrio parahaemolyticus OmpV (GenPept Accession No.BAC61661) 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＝256/258(99％)，Positives＝257/258(99％)，Gaps＝0/258(0％)
MNKTLIALLTLAAAGTATAGDTYIRNGNIYNNQGGWVAEVGVAQGSDLFKDQKHNTAPIL
Query 61 NGGYHGEDFNADLGGTNYRFLGETNDLINMSAYVVGSGIIRDGDVAKSLKGTQKRRLAVD 120
NGGYHGEDFNADLGG NYRFLGETNDLINMSAYVVGSGIIRDGDV+KSLKGTQKRRLAVD
Sbjct 61 NGGYHGEDFNADLGGINYRFLGETNDLINMSAYVVGSGIIRDGDVSKSLKGTQKRRLAVD 120
Query 121 LGLNTDFTLDEHNVISTYLQHDISGAYKGYLAGATYFHIMNFGSVDFVPFANLTYQSEDY 180
LGLNTDFTLDEHNVISTYLQHDISGAYKGYLAGATYFHIMNFGSVDFVPFANLTYQSEDY
Sbjct 121 LGLNTDFTLDEHNVISTYLQHDISGAYKGYLAGATYFHIMNFGSVDFVPFANLTYQSEDY 180
Query 181 VDYYFGIKDREATANRKAYKGDATVSYGLGYKLVMPINDNWQINQTTQYTRLXXXXXXXX 240
VDYYFGIKDREATANRKAYKGDATVSYGLGYKLVMPINDNWQINQTTQYTRLGSGISDSS
Sbjct 181 VDYYFGIKDREATANRKAYKGDATVSYGLGYKLVMPINDNWQINQTTQYTRLGSGISDSS 240
Query 241 XXXXANQWVVGASVSYNF 258
VVDSANQWVVGASVSYNF
Sbjct 241 VVDSANQWVVGASVSYNF 258
Above-listed: the proteic aminoacid sequence of Mermaid luminous bacillus OmpV
Following: the proteic aminoacid sequence of Vibrio parahaemolyticus OmpV (GenPept Accession No.BAC61439)
Therefore all there are higher homology in Mermaid luminous bacillus OmpV gene and Vibrio parahaemolyticus OmpV 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 Mermaid luminous bacillus OmpV
Below provide the variation of Mermaid luminous bacillus OmpV under the different salt concentration conditions:
Having extracted Mermaid luminous bacillus after the outer membrane protein under 0.5%, 1% and 4% these three 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 on amount gradually down gradually, this salt adaptability that shows this protein band and Mermaid luminous bacillus has the property of being closely related.Can know that in conjunction with qualification result this band is OmpV to Mermaid luminous bacillus outer membrane protein.
Behind proteic clone of Mermaid luminous bacillus OmpV and the abduction delivering to the influence of thalline brine sensitivity:
After by the method among the embodiment 1 Mermaid luminous bacillus OmpV 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 BL21OmpV transformant behind the IPTG abduction delivering.From Fig. 3, can be clear that the proteic expression of OmpV.Through the bacterial strain of abduction delivering and carried out the analysis of salt tolerance through inductive E.coli Top10+PLLP-OmpA-OmpV equally, table 3 is in each the observation on Growth result of salinity plating after 24 hours to this.
Table 3
| Salinity (1%) | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| E.coli BL21+PET | + | + | + | + | + | - | - |
| E.coli BL21+PET-OmpV | + | + | + | + | + | - | |
| E.coli Top10+PLLP-OmpA | + | + | + | + | + | - | - |
| E.coli Top10+PLLP-OmpA-OmpV | + | + | + | + | - | - | - |
Annotate :+expression has bacterium colony;-expression is not grown
As can be seen from Table 3, reduced a salinity on the tolerance of E.coli Top10+PLLP-OmpA-Omp comparison according to strain E.coli Top10+PLLP-OmpA salt, this has obviously shown Mermaid luminous bacillus OmpV and salt-sensitive directly related property.
Simultaneously, can see that E.coli BL21+PET-OmpV and its contrast strain E.coli BL21+PET are basic identical on the salt tolerance level, not show OmpV and import the decline of back its salt resistance ability.Its reason mainly is, still is present in the born of the same parents after OmpV expresses in E.coliBL21+PET-OmpV, is difficult to arrive the adventitia position, thereby can't brings into play its effect at brine sensitivity.And after using the PLLP-OmpA carrier since on the carrier with the effect of OmpA signal peptide, the OmpV albumen of abduction delivering can be secreted into outside the born of the same parents, OmpV albumen just shows its effect on brine sensitivity after inserting adventitia by certain mode.The Different Results of using these two kinds of carriers to draw further shown, OmpV expresses that to cause the lifting of brine sensitivity be the effect of OmpV albumen self, rather than causes other variation of cell behind its abduction delivering and produce.
The preparation and the purification of embodiment 4 Mermaid luminous bacillus OmpV polypeptide
The Mermaid luminous bacillus OmpV 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 Mermaid luminous bacillus OmpV 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 in the cloning vector E.coli DH5 α competence with chemical heat shock method.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 Mermaid luminous bacillus OmpV 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 PCR respectively verify, obtain PCR checking 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 Mermaid luminous bacillus OmpV 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 hour for 37 ℃.
Express screening, the evaluation of the proteic BL21 positive colony of Mermaid luminous bacillus OMPV:
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 IPTG100ug/ml induced two hours for 35 ℃, 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 OmpV albumen in transgenosis E.coli:
1, proteic obtaining: express Mermaid luminous bacillus OmpV 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 pH6.8 1ml; 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-Mermaid luminous bacillus OmpV), 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.
Sequence table
＜110〉Zhongshan University
＜120〉Mermaid luminous bacillus outer membrane protein V and encoding sequence and its production and application
<140>200510100213.2
<141>2005-10-11
<160>4
<210>1
<211>22
<212>DNA
＜213〉artificial sequence primer
<400>1
GGGGATCCATGAACAAGACACT
<210>2
<211>26
<212>DNA
＜213〉artificial sequence primer
<400>2
CCGCTAGCTTAGAAGTTGTAAGACAC
<210>3
<211>777
<212>DNA
＜213〉Mermaid luminous bacillus (Photobacterium damsela)
<400>3
atgaacaaga cacttatcgc actactaact ttggcagcag ctggcaccgc aacggcaggt 60
gacacataca tccgtaacgg caacatctac aacaaccaag gtggttgggt tgcggaagtg 120
ggcgtagctc aaggtagtga cctatttaaa gatcaaaaac acaatactgc accaatccta 180
aacggtggtt accacggtga agacttcaac gcagatttgg gcggcaccaa ctaccgcttc 240
ttgggtgaaa ccaacgatct gatcaacatg agcgcatacg tggtaggcag cggcattatt 300
cgtgatggcg atgtagcaaa gagcttgaaa ggcacacaaa aacgtcgttt agcggttgat 360
ttaggtctaa ataccgattt cactctagat gagcacaacg tcatctcgac atacctacaa 420
cacgacatca gcggcgcata caaaggttac ttagcgggcg cgacgtactt ccacatcatg 480
aacttcggta gtgttgattt tgttcctttt gctaacctaa cttatcaaag tgaagactac 540
gtagattact actttggcat caaagatcgt gaagcgacgg caaaccgcaa agcatacaaa 600
ggtgatgcaa cggtaagcta cggtctgggc tacaagctgg ttatgccaat taatgacaac 660
tggcaaatca accaaacaac tcaatacact cgcctaggca gtggtatcag tgattcatct 720
gttgtagata gcgcaaacca atgggttgtt ggcgcaagcg tgtcttacaa cttctaa 777
<210>4
<211>258
<212>PRT
＜213〉Mermaid luminous bacillus (Photobacterium damsela)
<400>4
Met Asn Lys Thr Leu Ile Ala Leu Leu Thr Leu Ala Ala Ala Gly Thr
1 5 10 15
Ala Thr Ala Gly Asp Thr Tyr Ile Arg Asn Gly Asn Ile Tyr Asn Asn
20 25 30
Gln Gly Gly Trp Val Ala Glu Val Gly Val Ala Gln Gly Ser Asp Leu
35 40 45
Phe Lys Asp Gln Lys His Asn Thr Ala Pro Ile Leu Asn Gly Gly Tyr
50 55 60
His Gly Glu Asp Phe Asn Ala Asp Leu Gly Gly Thr Asn Tyr Arg Phe
65 70 75 80
Leu Gly Glu Thr Asn Asp Leu Ile Asn Met Ser Ala Tyr Val Val Gly
85 90 95
Ser Gly Ile Ile Arg Asp Gly Asp Val Ala Lys Ser Leu Lys Gly Thr
100 105 110
Gln Lys Arg Arg Leu Ala Val Asp Leu Gly Leu Asn Thr Asp Phe Thr
115 120 125
Leu Asp Glu His Asn Val Ile Ser Thr Tyr Leu Gln His Asp Ile Ser
130 135 140
Gly Ala Tyr Lys Gly Tyr Leu Ala Gly Ala Thr Tyr Phe His Ile Met
145 150 155 160
Asn Phe Gly Ser Val Asp Phe Val Pro Phe Ala Asn Leu Thr Tyr Gln
165 170 175
Ser Glu Asp Tyr Val Asp Tyr Tyr Phe Gly Ile Lys Asp Arg Glu Ala
180 185 190
Thr Ala Asn Arg Lys Ala Tyr Lys Gly Asp Ala Thr Val Ser Tyr Gly
195 200 205
Leu Gly Tyr Lys Leu Val Met Pro Ile Asn Asp Asn Trp Gln Ile Asn
210 215 220
Gln Thr Thr Gln Tyr Thr Arg Leu Gly Ser Gly Ile Ser Asp Ser Ser
225 230 235 240
Val Val Asp Ser Ala Asn Gln Trp Val Val Gly Ala Ser Val Ser Tyr
245 250 255
Asn Phe
Claims (7)
1, Mermaid luminous bacillus outer membrane protein V is characterized in that: this proteic aminoacid sequence is shown in SEQ ID NO.4.
2, a kind of isolating polynucleotide, it is characterized in that: this polynucleotide sequence is selected from down group:
(a) polynucleotide of aminoacid sequence according to claim 1 of encoding;
(b) with polynucleotide (a) complementary polynucleotide.
3, polynucleotide as claimed in claim 2 is characterized in that: the aminoacid sequence shown in this polynucleotide encoding SEQ ID NO.4.
4. polynucleotide as claimed in claim 2 is characterized in that: the sequence of these polynucleotide is selected from coding region sequence or the full length sequence of SEQ ID NO.3.
5, the preparation method of Mermaid luminous bacillus outer membrane protein V is characterized in that comprising following steps:
(1) will the encode nucleotide sequence of the described Mermaid luminous bacillus outer membrane protein V of claim 1 operationally is connected in expression regulation sequence, forms the Mermaid luminous bacillus outer membrane protein V expression vector;
(2) change the expression vector in the step (1) over to host cell, form the reconstitution cell of Mermaid luminous bacillus outer membrane protein V;
(3) be fit to express under the condition of Mermaid luminous bacillus outer membrane protein V polypeptide, the reconstitution cell in the culturing step (2) is expressed target product;
(4) separation and purification Mermaid luminous bacillus outer membrane protein V.
6, Mermaid luminous bacillus outer membrane protein V as claimed in claim 1 is in the application that improves the biomass cells brine sensitivity.
7, polynucleotide as claimed in claim 2 are in the application that improves the biomass cells brine sensitivity.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO1998038315A1 (en) * | 1997-02-28 | 1998-09-03 | Japan Tobacco Inc. | GENE ENCODING β-GALACTOSIDE α-2,6-SIALYLTRANSFERASE |
Non-Patent Citations (1)
| Title |
|---|
| Vibrio parahaemolyticus outer membrane protein (ompV)gene,Vibrio parahaemolyticus outer membrane protein (ompV)gene,complete cds. Mao,Z.J.,Chu,W.Y. and Zhang,C.W,GeneBank 2005 * |
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