CN101191133B - Carrier for catching secretion sequence and its construction method and application thereof - Google Patents

Abstract

The invention relates to the molecular biology and genetic engineering field, in particular to a carrier for capturing secretion sequence and a constructive method and application of the carrier. The capturing carrier contains agar enzyme genes agaV excluded signal sequence, an exogenous gene insertion sites and filtered markings. The mature agar enzyme genes agaV is utilized to be taken as reportmolecules, and functional signal sequences and secretory protein genes can be quickly and highly efficiently filtered from Gram-positive bacteria and Gram-negative bacteria. The invention not only ca n filter the secretory protein genes from unknown genomes but also can verify presumptive secretory signals, detect functions of promoters and SD sequences, and extensively filter manual strong force signal peptides and so on. Moreover, operation of the invention is simple and only conventional connection, conversion and color filtration are needed; captured genes have no false positive and 100 percent of the captured genes have typical characteristics of the secretory protein genes, thereby the invention provides candidate vaccine antigens and medicine targets for prevention and cure of pathogenic microorganism diseases.

Description

A kind of carrier of catching secretion sequence and construction process thereof and application

Technical field

The present invention relates to molecular biology and genetically engineered field, specifically a kind of carrier of catching secretion sequence and construction process thereof and application.

Background technology

Protein one of the basic support that exists of relying for all life systems.In various cell proteins, secreted protein (comprising cytolemma guidance quality albumen and pericentral siphon chamber albumen) is an important class.These albumen contain the signal that can be discerned by the emiocytosis factor usually in its primary structure, Jing Dian I type secretory protein for example, especially the excretory albumen via the Sec approach, usually contain a signal peptide of being made up of 20-35 amino acid at its N end, the proteic formation of successfully striding film transportation and functional structure picture that this signal peptide draws for its rate has decisive role.Based on its particular structure and effect characteristics, secreted protein has important biomolecule function usually in prokaryotic organism, for example to the perception and the conduction of ambient signal, interact with host cell, population density induction etc., these unique functions make secreted protein Chang Zuowei medicine and vaccine target spot in the pathogenic micro-organism diseases prevention and treatment, thereby the screening of the systematicness of secretory protein is significant for the life mechanism of research bacterium, pathogenesis, disease control etc.

Agarase is a kind of natural glycoside hydrolase, how is produced by marine microorganism, and its effect substrate is an agar-agar, and 1,3 β-D-or 1,4 α-L-galacto-pyranose in can the cracking agar-agar connect.Though various in recent years novel product agarase bacterial strains constantly are found, agarase gene and proteic research also day by day deeply, yet it is few really to be successfully applied to the agarase of industry or scientific research field.The actual property application of agarase at present mainly is limited to reclaims DNA or other molecule from the agar-agar embedding medium, its potentiality is used and is comprised and be used to prepare the agar-agar oligosaccharide etc. of deriving, but the research that the agarase gene is used as a kind of biology tool does not appear in the newspapers as yet.

Agarase agaV is a conservative property I class secretory protein, have typical Sec-translocon dependent form secretory protein constitutional features and matrix folding ability of mind efficiently, and outer active the detection simply directly of its born of the same parents, yet, the report that utilizes agarase gene trap secretion signal is not arranged up to now in the world as yet.

Summary of the invention

The object of the invention is a kind of carrier and construction process and application of advantages of simplicity and high efficiency catching secretion protein gene sequence.

For achieving the above object, technical scheme of the present invention is: carrier is: have base sequence among the sequence table SEQ IDNo:2, contain the agarase gene agaV of the number of writing to sequence, foreign gene inserts site and selection markers.

Construction process:

1) design plasmid pBK:

A. be template with plasmid pACYC177,, use restriction enzyme BamHI and HincII double digestion behind the PCR product purification with the kanamycin gene primer that contains BamHI site and HincII site respectively, pcr amplification kanamycin gene;

Wherein: the PCR condition is: 94 ℃ of pre-sex change template DNAs of 60s, 94 ℃ of 40s then, 45 ℃ of 60s, 72 ℃ of 60s change 94 ℃ of 40s into after 5 circulations, 58 ℃ of 60s, 72 ℃ of 60s, after 25 circulations again at 72 ℃ of extension 10min;

B. plasmid pBR322 is cut with BamHI and ScaI enzyme, electrophoresis reclaims the 3471bp dna fragmentation, cut the PCR product of handling and be connected obtaining enzyme in this fragment and a step, connect liquid transformed into escherichia coli DH5 α, incubated overnight in containing kantlex LB substratum, filter out the transformant of anti-kantlex, from transformant, extract recombinant plasmid, be pBK;

2) design plasmid pUALS:

A. the operon of agaV is cloned into plasmid pUC19, constitute recombinant plasmid pBUA1, be template then with plasmid pBUA1, primer with containing BamHI site and XhoI site respectively carries out pcr amplification, obtains the PCR product of the agaV of the number of writing to sequence, with the PCR product purification, then be connected in room temperature, connect mixed solution transformed into escherichia coli DH5 α, on the LB solid medium that contains 100ug/ml Ap, 40ug/ml Xga1 and 24ug/ml IPTG, cultivate the white transformant of screening with carrier pBS-T;

Wherein: the PCR condition: 94 ℃ of pre-sex change template DNAs of 60s, 94 ℃ of 40s then, 46 ℃ of 60s, 72 ℃ of 60s change 94 ℃ of 40s into after 5 circulations, 59 ℃ of 60s, 72 ℃ of 60s, after 25 circulations again at 72 ℃ of extension 10min;

B. from the white transformant of picking, extract recombinant plasmid, be pUALS;

3) design secretion sequence capturing carrier:

A. the plasmid pBK that obtains in the step 1) is cut with restriction enzyme BamHI and BsaBI enzyme, obtain the 3.2kbDNA fragment; With step 2) in plasmid pUALS with restriction enzyme BamHI and SmaI double digestion, obtain the 1.3kb dna fragmentation, it is connected with preceding 3.2kb dna fragmentation, connects and containing the transformant that screens anti-kantlex on the LB solid medium of Kn after liquid is transformed into bacillus coli DH 5 alpha;

B. from the transformant of anti-kantlex, extract recombinant plasmid, be secretion sequence capturing carrier pBU.

Primer among the described step 1) a is KnF2 5 '-AT GGATCCACGGTTGATGAGAGC-3 ', KnR3 5 '-GCC GTCGACCAATTAACCAATTC-3 '.

Described step 2) primer among a is UAF5 5 '-AT GGATCCGAAGACTGGCGAGAAATC-3 ' UAR3 5 '-CA CTCGAGTTGTATAAATTTCAATCGC-3 '.

Described step 2) obtain the PCR product purification of agaV of the number of writing to sequence after, be connected 2-4 hour with carrier pBS-T in room temperature.

Described carrier can be caught secreted protein gene in the prokaryotic organism.

The present invention has following advantage:

1. applied widely.The present invention can be used for the screening of prokaryotic organism signal sequence, especially the still unknown microorganism of genome sequence.

2. functional diversity.The present invention not only can screen secreted protein gene from the unknown gene group, but also can be to the Function detection of checking, promotor and SD (ShineDalgarno) sequence of supposition (putative) secretion signal, the extensive screening of artificial strong signal peptide etc.

3. simple high efficiency.The present invention is simple to operate, only needs conventional connection, conversion and dithering; The gene non-false positive of being caught, 100% has typical secreted protein gene feature.

Description of drawings

Fig. 1 makes up the schema of catching secretion protein gene sequence carrier for the present invention.

Representative S.iniae (G26) the protein signal peptide prognostic chart that Fig. 2 the present invention filters out.

Representative V.harveyi (T4) the protein signal peptide prognostic chart that Fig. 3 the present invention filters out.

Embodiment

The invention will be further described below in conjunction with drawings and Examples.Embodiment is intended to the description of giving an example to the present invention, but not limits the invention in any form.

The present invention utilizes agaV structure gene as reporter gene, becomes a kind of secretion signal capturing carrier with the plasmid pBR322 plasmid construction of deriving; In this capturing carrier, foreign DNA can be inserted into the upstream of agaV gene via a BamHI site; Because disappearance signal sequence and promotor, agaV gene in this capturing carrier is beyond expression, if but contain a functional gene in the exogenous dna fragment that inserts in coding region fracture (truncated), and 5 ' coding region of this split gene and agaV gene are connected to form in-frame and merge, and the mode that the agaV gene just can fusion gene is expressed so; If the split gene that inserts contains secretory signal sequence, and this signal sequence can be discerned by colibacillary excretory system, the heterologous peptides of Rong Heing is tolerated by agaV simultaneously, do not influence the formation and the functionally active in its key structure territory, the form that the agaV after expressing so then can fusion rotein be transported to born of the same parents outer and have with wild-type similarly, can detected activity.The split gene sequence of being inserted can be known via the agaV upstream is checked order, use methods such as chromosome walking, molecular hybridization to clone at an easy rate subsequently and obtain complete secretory gene.Because the conservative property and the extensive existence of I class film guiding protein in prokaryotic organism of Sec excretory system, this capturing carrier can be applied to the screening of all prokaryotic organism (comprising Gram-positive and gram negative bacterium) secretion signal, and thus obtained secretory protein can be used for multiple purpose.

Involved in embodiments of the present invention experimental technique all adopts following method:

1. plasmid extraction, PCR product purification, corresponding reagent box (the Plasmid Mini Kit I that dna fragmentation reclaims from gel, the bacterial genomes DNA extraction is all used U.S. Omega Bio-Tek company, Cycle-pure Kit, Gel Extraction Kit, Bacterial DNA Kit)

2. plasmid, the conversion of DNA connection liquid enter intestinal bacteria and all use Hanaban method (Sambrook andRussell:Molecular Cloning:A Laboratory Mannual.Cold Spring HarborLaboratory Press 2001);

3. all restriction enzymes and ligase enzyme are all available from " Niu Yinglun Bioisystech Co., Ltd ", Beijing.

Embodiment 1

The building process (referring to Fig. 1) of secretion signal capturing carrier pBU

The structure of step 1) carrier pBK.

A. be template with plasmid pACYC177 (coming from " Niu Yinglun Bioisystech Co., Ltd ", Beijing), with following primer PCR kantlex (Kn) gene: KnF2 (5 '-AT GGATCCACGGTTGATGAGAGC-3 ', the line base is the BamHI site), KnR3 (5 '-GCC GTCGACCAATTAACCAATTC-3 ', the line base is the HincII site), the PCR condition is: 94 ℃ of pre-sex change template DNAs of 60s, 94 ℃ of 40s then, 45 ℃ of 60s, 72 ℃ of 60s change 94 ℃ of 40s into after 5 circulations, 58 ℃ of 60s, 72 ℃ of 60s, after 25 circulations again at 72 ℃ of extension 10min.The PCR product is used restriction enzyme BamHI and HincII double digestion after using Omega Cycle-pure test kit purifying, then with using Omega Cycle-pure test kit purifying.

B. with plasmid pBR322 (available from " Shanghai Sangon Biological Engineering Technology And Service Co., Ltd ", Shanghai) cut with BamHI and ScaI enzyme, electrophoresis reclaims the 3471bp dna fragmentation, this fragment and preceding BamHI/HincII enzyme are cut the PCR product of handling to be connected, connect liquid Hanahan method transformed into escherichia coli DH5 α, on the Luria-Bertani that contains Kn (50ug/ml) (LB) culture medium flat plate, screen transformant, extract the plasmid of transformant with Omega Plasmid Mini Kit, be plasmid pBK, this plasmid after restriction enzyme BamHI and SmaI enzyme are cut in 0.8% sepharose electrophoresis, electrophoresis result shows that it is two bands that enzyme is cut product, article one, size is about 550bp, another is 4kb, illustrates that this plasmid is correct recombinant plasmid.

Described LB moiety is by weight percentage: 1.0% peptone, 0.5% yeast powder, 1.0% sodium-chlor.

Step 2) structure of carrier pUALS.

A. the operon of the agarase agaV in the Vibrio anguillarum being cloned into plasmid pUC19 (commercial), constituting recombinant plasmid pBUA1, is template with plasmid pBUA1 then, with primer UAF5 (5 '-AT GGATCCGAAGACTGGCGAGAAATC-3 ', the line base is the BamHI site) and UAR3 (5 '-CA CTCGAGTTGTATAAATTTCAATCGC-3 ', the line base is the XhoI site) carry out pcr amplification, obtain the PCR product of the agaV of the number of writing to sequence.The PCR product with Omega Cycle-pure test kit purifying after with PCR cloning vector pBS-T (" day root biochemical technology company limited ", Beijing) connect 2 hours in room temperature, connect mixed solution with Hanahan method transformed into escherichia coli DH5 α after cultivation 18 hours on the LB solid medium that contains 100ug/ml Ap, 40ug/ml Xga1 and 24ug/ml IPTG, filter out white transformant.

The PCR condition: 94 ℃ of pre-sex change template DNAs of 60s, 94 ℃ of 40s then, 46 ℃ of 60s, 72 ℃ of 60s change 94 ℃ of 40s into after 5 circulations, 59 ℃ of 60s, 72 ℃ of 60s, after 25 circulations again at 72 ℃ of extension 10min.

Wherein agarase agaV sequence table is as follows:

atgaaatcca?taattaaaac?tctgacattg?ctaccaatat?ttttaactag?cacgacgtta

atggctgaag?actggcgaga?aatccccatt?ccagcaccgt?tagaagatgg?gcagagctgg

gagttacaag?aggcatactc?cgattcgttt?aattatacgg?gtaagggcga?ctctttccgt

agtaagtgga?atgataccta?ctttcacggt?tggacaggac?ctggtttaac?ctattggcaa

agtgatgagt?catgggtaga?tgatggcaac?ttgataatca?gtgcctctcg?tcgtcaggga

acagagatgg?tgaatgcggg?cgttgtgacg?tctaagacaa?aggtgaaata?tcctattttt

ctagaggcca?atataaaggt?gagtggctta?gagctatcat?caaatttttg?gcttcttagt

gaaaatgatc?aacgagaaat?cgatgtattg?gaagtgtatg?gtggcgctga?agacgaatgg

tttgccaaaa?atatgtcaac?aaacttccat?gtatttttcc?gcaatgaaga?caactcaatt

cgtagcgatt?tcaacgatca?gacccacaat?gagccgcaat?ggggtactta?ttggcgtgat

gggttccatc?gctttgcggt?atattggaaa?agcccaactg?aagtgacttt?ttatatcaat

ggcatagaga?caccgaaagg?ttcttgggaa?gacgtcgtaa?tgaaagataa?agattacacg

ggagccatcc?tagataaatc?aacctacaac?atggatcaag?agatgttcat?tattattgat

actgaagacc?attcgtggcg?ttctgaggct?ggaataatcg?caaaagatga?agacctagct

gatgatagta?agaacaaaat?gtatgtggat?tggatacgtg?tttataagcc?agttggcgga

gatgacaatg?gtaatgtgac?cgtaccgtca?gggtatacaa?acacacaatt?tgtgcatagt

gacttgtgtt?tggatgtcaa?agatggagcg?acttggaacg?gcagtactta?tcagcaatgg

gtgtgtaata?cgggcaacct?gaaccaacga?tttgggtttg?aagaagtcgc?gacgggagag

tatttaatta?aatccaaacg?tagtcagctg?tgcttagagc?tcaagcccgg?tagtaatcaa

tggcaggacg?gtgcgatcat?tcagcaatat?gtgtgtaatt?cagctgaaga?gaatcaacgt

tggacgctat?ttgataaagg?cagccagacg?tttgagattc?gaagcaaggc?aacaggtaag

tgtttagagc?ttgctgacaa?tcagggaagc?aacggcggga?cagtgcagca?atggtcatgt

gatggtggta?ataaccagcg?attgaaattt?atacaataa

B. adopt Omega Plasmid Mini Kit to extract plasmid in the white transformant, be plasmid pUALS, this plasmid with restriction enzyme BamHI/XhoI double digestion after in 0.8% sepharose electrophoresis, electrophoresis result shows that it is two bands that enzyme is cut product, article one, size is about 1.3kb (agaV inserts fragment), another is 3kb (a pBS-T carrier), illustrates that this plasmid is correct recombinant plasmid.

The structure of step 3) secretion sequence capturing carrier pBU.

The plasmid pBK of step 1) is cut with restriction enzyme BamH I and BsaBI enzyme, and electrophoresis in 0.8% sepharose reclaims the 3.2kb dna fragmentation; With step 2) plasmid pUALS cut with restriction enzyme BamHI and SmaI enzyme, electrophoresis in 0.8% sepharose, reclaim the 1.3kb dna fragmentation, it is connected with 3.2kb pBK/BamHI/BsaBI fragment, connects and on the LB solid medium that contains Kn (50ug/ml), screen transformant after liquid is transformed into bacillus coli DH 5 alpha with the Hanahan method.2 transformants of picking, extract plasmid with Omega Plasmid Mini Kit, be plasmid pBU (referring to sequence table 2), this plasmid with restriction enzyme BamHI/XhoI double digestion after in 0.8% sepharose electrophoresis, electrophoresis result shows that it is two bands that enzyme is cut product, article one, size is about 1.3kb, and another is 3.2kb, illustrates that this plasmid is correct recombinant plasmid, pBU total length: 4577bp, be double-stranded DNA, pBU characteristics (main element): agaV:1-1302, Kn ^r: 4577-3480, Origin:2773-2185.

Embodiment 2

Adopt capturing carrier pBU to screen secretion sequence and secreted protein gene (G26) from gram-positive microorganism Streptococcus iniae (Streptococcus iniae).(its composition is: peptone 1.7% at 5ml TSAYE substratum, polypeptone 0.3%, yeast extract 0.6%, NaCl 1.5%, K2HPO40.327%, glucose 0.25%, agar 1.5%) middle incubated overnight S.iniae bacterial strain G26, extract genomic dna with Omega Bacterial DNA test kit, with the 5ug genomic dna partially digested back of restriction enzyme Sau3AI electrophoresis in 0.8% agar gel, reclaim the 4-6kb dna fragmentation with Omega GelExtraction test kit, the DNA that 400ng is reclaimed and 200ngBamHI linearizing and the plasmid pBU that handled through calf alkaline phosphatase (CIP) are connected, and the connection mixed solution screens transformant after with Hahahan method transformed into escherichia coli DH5 α on the LB nutrient agar that contains Kn (50ug/ml).From transforming on the flat board transformant of 6 peripheral substratum depressions of picking at random, with transformant incubated overnight in the LB substratum that contains Kn (50ug/ml) of picking, extract plasmid and its contained insertion dna fragmentation is checked order with primer UAR6 (5 '-CTCATCACTTTGCCAATAGG-3 ') with OmegaPlasmid Mini Kit test kit.Sequencing result finds all to contain in all 6 recombinant plasmids the fracture functional gene of a tool signal peptide sequence behind bioinformatic analysis as follows, wherein: signal peptide is made up of about 15-35 amino acid usually, be positioned at the N end or the C end of secretory protein, it is formed amino acid and mostly is hydrophobicity and nonpolar amino acid, but near the several amino acid its N end is generally basic aminoacids, and line amino acid is signal peptide characteristic strong basicity amino acid in the bracket.

BUG1

(MF

ET

TLL

SPNLWVTIIGIALVPALYCLVFLSSMWNPYA)HFNQ

FPVAVVNQDKGASLDNKSINVGNEMVDTLSKNKDLDFHFVSEKTAQEK

LDAGKYYMIITFPDNFSSKVASVM

BUG2

(MG

SYQLKKTSSKGFKLTVLSFVLLTLLVSVWSLKKA)RQ

DLKPLAETASLSFIRKSAHSAADIARKNDLYTSVMLAQATLESQNGQSQLSQKPY

YNFFGVKGDYKGKSAVLPTLEDDGQGNLYTVDAKFRSYGKMINSFKD

YATVLSDPLYENTHKAKTKHYQEATATLTGRYATDTSYHVKLNNLIET

YRLYYFD

It is as follows that its coded product of fracture functional gene of catching is all secreted protein: wherein the capitalization part is secretory signal sequence in this sequence

G26

BUG1：

ATGTTTAAAGAAACAAGGACATTATTAAAAAGCCCAAACCTATGGGTAACAATCATAGGGATAG

CTTTGGTTCCTGCACTATATTGCCTGGTTTTCCTCAGTTCTATGTGGAATCCTTATGCCcacttcaaccaa

tttccagttgccgttgtcaatcaagataagggtgctagcttagataataaatccatcaacgtcggaaatgagatggtggacacactgtcaaaaaataaggactt

ggattttcatttcgtttctgaaaaaacagctcaagaaaaacttgatgctgggaaatactatatgattatcactttcccagataatttttcaagtaaagttgcctctgtg

atgac

BUG2：

ATGGGCAGAAAAAAAAGAAAAAGAAGTTATCAGCTAAAAAAAACAAGTTCAAAAGGGTTTAA

GTTAACTGTTTTAAGTTTTGTTCTTTTGACATTACTAGTTTCAGTATGGTCTTTAAAAAAAGCTagg

caagatttgaaacctttagctgaaacagcctctttatcatttataagaaaaagtgcacattcagcggcagatatagcaagaaaaaatgatctatatacgtcagtg

atgcttgcgcaagcaactctagagtcgcaaaatggccaatcccaattgagtcaaaaaccttattataatttctttggggtgaaaggagattacaaaggaaaatc

tgctgttttacccactctagaagatgatggtcaagggaatctttatacagttgatgcaaagtttagatcttacggaaaaatgattaattcttttaaagactatgcaac

tgtattatcagaccctttgtatgaaaatactcataaagctaaaacaaaacactatcaagaagcaacagcaactttaacgggtcgttatgcaacagacacgtctta

tcatgtcaaattaaataatcttattgagacttataggctttattattttgacta

Can get the sequence (referring to Fig. 2) that secretion sequence that carrier that the present invention makes up catches is all the coding secreted protein by detecting.

Embodiment 3

(its composition is: peptone 1.7% at the 5mlTSAYE substratum from Gram-negative bacteria Vibrio harveyi V.harveyi (T4) to adopt capturing carrier pBU, polypeptone 0.3%, yeast extract0.6%, NaCl 1.5%, K2HPO4 0.327%, glucose 0.25%, agar 1.5%) middle incubated overnight V.harveyi bacterial strain T4, extract genomic dna with Omega Bacterial DNA test kit, with the 5ug genomic dna partially digested back of restriction enzyme Sau3AI electrophoresis in 0.8% agar gel, reclaim the 4-6kb dna fragmentation with Omega Gel Extraction test kit, the DNA that 400ng is reclaimed and 200ng BamHI linearizing and the plasmid pBU that handled through calf alkaline phosphatase (CIP) are connected, and the connection mixed solution screens transformant after with Hanahan method transformed into escherichia coli DH5 α on the LB nutrient agar that contains Kn (50ug/ml).From transforming on the flat board transformant of 7 peripheral substratum depressions of picking at random, with transformant incubated overnight in the LB substratum that contains Kn (50ug/ml) of picking, extract plasmid and its contained insertion dna fragmentation is checked order with primer UAR6 (5 '-CTCATCACTTTGCCAATAGG-3 ') with Omega Plasmid Mini Kit test kit.Sequencing result finds all to contain in all 7 recombinant plasmids the fracture functional gene of a tool signal peptide sequence behind bioinformatic analysis as follows, wherein: signal peptide is made up of about 15-35 amino acid usually, be positioned at the N end or the C end of secretory protein, it is formed amino acid and mostly is hydrophobicity and nonpolar amino acid, but near the several amino acid its N end is generally basic aminoacids, and line amino acid is signal peptide characteristic strong basicity amino acid in the bracket.

BUS1

(M

TIAAALLACLSFSANA)AEYRANEVANGFQIPWGIEFLDNQRVI

VNEKNGTVSLLDIKSGKPQKLFNVPDVNTSGQAGLLDVALTPNADKQ

NPQLFFTYSKRTSK

BUS2

(M

ILLFSILAYSTLSYA)RIPNSLQDQNITITVKYDKETQEFYDLSATG

SNYFNYQIRNDGFEITHGRDGFIGASTEFNNQSIRVGLDTNQDLGVKY

LINTSTGAKASLDVGLLSAFFRYQGQSANAHSDNAQN

It is as follows that its coded product of fracture functional gene of catching is all secreted protein: wherein the capitalization part is screening secretion sequence and secreted protein gene in the secretory signal sequence in this sequence.

T4

BUS1：

ATGAGAACGATTGCTGCTGCCCTACTCGCTTGTTTATCCTTTTCTGCCAACGCTgccg

aataccgcgctaacgaagtggcgaatggatttcagatcccttggggaatcgagttcttagacaaccaacgcgttatcgtcaatgaaaag

aacggtacggtttcgctgctggatatcaaatcaggcaaacctcaaaagctgtttaacgtccccgatgtgaacaccagtggacaagcgg

gcttattggatgttgcgctcacaccgaatgccgataagcaaaacccgcaactcttctttacctacagcaagcgtacgagcaaagg

BUS2-Blue：

ATGAAAAAAATATTATTGTTTTCGATACTTGCTTACTCAACACTTAGTTACGCAagaatc

cccaattctctgcaagaccagaatattacaattactgtaaagtatgataaggaaactcaagaattctatgatttgagtgctactggatccaat

tattttaactatcagataagaaacgatggttttgaaataacacatggtcgagatgggttcataggagcgagtacagagtttaataaccaatc

tataagggttgggctagatacgaatcaagacctaggtgtaaagtacttaattaacacgtcgacaggagcaaaagcatcgttggatgttgg

tcttttaagtgctttctttaggtatcaaggacagtcagctaatgctcacagcgacaacgcacagaatt

Can get the sequence (referring to Fig. 3) that secretion sequence that carrier that the present invention makes up catches is all the coding secreted protein by detecting.

Embodiment 2 and embodiment 3 results show that pBU can be used as a kind of broad spectrum (general) screening implement, screening function secretion sequence and secreted protein gene from Gram-positive and Gram-negative bacteria fast and efficiently.

The embodiment of the invention is intended to the description of giving an example to the present invention, but not limits the invention in any form.

(1) information of SEQ ID No:2 (referring to sequence table)

(a) sequence signature

* length: 4577 base pairs

* type: nucleic acid

* chain: two strands

* topological framework: ring-type

(b) molecule type: DNA

(c) suppose: not

(d) antisense: not

(e) initial source: synthetic

Sequence table

SEQUENCE?LISTING

＜110〉Institute of Oceanology of the Chinese Academy of Sciences

＜120〉a kind of carrier of catching secretion sequence and construction process thereof and application

<130>

<160>2

<170>PatentIn?version?3.1

<210>1

<211>1359

<212>DNA

<213>Vibrio?sp.

<220>

<221>gene

<222>(1)..(1359)

<223>

<400>1

atgaaatcca?taattaaaac?tctgacattg?ctaccaatat?ttttaactag?cacgacgtta 60

atggctgaag?actggcgaga?aatccccatt?ccagcaccgt?tagaagatgg?gcagagctgg 120

gagttacaag?aggcatactc?cgattcgttt?aattatacgg?gtaagggcga?ctctttccgt 180

agtaagtgga?atgataccta?ctttcacggt?tggacaggac?ctggtttaac?ctattggcaa 240

agtgatgagt?catgggtaga?tgatggcaac?ttgataatca?gtgcctctcg?tcgtcaggga 300

acagagatgg?tgaatgcggg?cgttgtgacg?tctaagacaa?aggtgaaata?tcctattttt 360

ctagaggcca?atataaaggt?gagtggctta?gagctatcat?caaatttttg?gcttcttagt 420

gaaaatgatc?aacgagaaat?cgatgtattg?gaagtgtatg?gtggcgctga?agacgaatgg 480

tttgccaaaa?atatgtcaac?aaacttccat?gtatttttcc?gcaatgaaga?caactcaatt 540

cgtagcgatt?tcaacgatca?gacccacaat?gagccgcaat?ggggtactta?ttggcgtgat 600

gggttccatc?gctttgcggt?atattggaaa?agcccaactg?aagtgacttt?ttatatcaat 660

ggcatagaga?caccgaaagg?ttcttgggaa?gacgtcgtaa?tgaaagataa?agattacacg 720

ggagccatcc?tagataaatc?aacctacaac?atggatcaag?agatgttcat?tattattgat 780

actgaagacc?attcgtggcg?ttctgaggct?ggaataatcg?caaaagatga?agacctagct 840

gatgatagta?agaacaaaat?gtatgtggat?tggatacgtg?tttataagcc?agttggcgga 900

gatgacaatg?gtaatgtgac?cgtaccgtca?gggtatacaa?acacacaatt?tgtgcatagt 960

gacttgtgtt?tggatgtcaa?agatggagcg?acttggaacg?gcagtactta?tcagcaatgg 1020

gtgtgtaata?cgggcaacct?gaaccaacga?tttgggtttg?aagaagtcgc?gacgggagag 1080

tatttaatta?aatccaaacg?tagtcagctg?tgcttagagc?tcaagcccgg?tagtaatcaa 1140

tggcaggacg?gtgcgatcat?tcagcaatat?gtgtgtaatt?cagctgaaga?gaatcaacgt 1200

tggacgctat?ttgataaagg?cagccagacg?tttgagattc?gaagcaaggc?aacaggtaag 1260

tgtttagagc?ttgctgacaa?tcagggaagc?aacggcggga?cagtgcagca?atggtcatgt 1320

gatggtggta?ataaccagcg?attgaaattt?atacaataa 1359

<210>2

<211>4577

<212>DNA

＜213〉synthetic

<220>

<221>gene

<222>(1)..(4577)

<223>

<400>2

ggatccgaag?actggcgaga?aatccccatt?ccagcaccgt?tagaagatgg?gcagagctgg 60

gagttacaag?aggcatactc?cgattcgttt?aattatacgg?gtaagggcga?ctctttccgt 120

agtaagtgga?atgataccta?ctttcacggt?tggacaggac?ctggtttaac?ctattggcaa 180

agtgatgagt?catgggtaga?tgatggcaac?ttgataatca?gtgcctctcg?tcgtcaggga 240

acagagatgg?tgaatgcggg?cgttgtgacg?tctaagacaa?aggtgaaata?tcctattttt 300

ctagaggcca?atataaaggt?gagtggctta?gagctatcat?caaatttttg?gcttcttagt 360

gaaaatgatc?aacgagaaat?cgatgtattg?gaagtgtatg?gtggcgctga?agacgaatgg 420

tttgccaaaa?atatgtcaac?aaacttccat?gtatttttcc?gcaatgaaga?caactcaatt 480

cgtagcgatt?tcaacgatca?gacccacaat?gagccgcaat?ggggtactta?ttggcgtgat 540

gggttccatc?gctttgcggt?atattggaaa?agcccaactg?aagtgacttt?ttatatcaat 600

ggcatagaga?caccgaaagg?ttcttgggaa?gacgtcgtaa?tgaaagataa?agattacacg 660

ggagccatcc?tagataaatc?aacctacaac?atggatcaag?agatgttcat?tattattgat 720

actgaagacc?attcgtggcg?ttctgaggct?ggaataatcg?caaaagatga?agacctagct 780

gatgatagta?agaacaaaat?gtatgtggat?tggatacgtg?tttataagcc?agttggcgga 840

gatgacaatg?gtaatgtgac?cgtaccgtca?gggtatacaa?acacacaatt?tgtgcatagt 900

gacttgtgtt?tggatgtcaa?agatggagcg?acttggaacg?gcagtactta?tcagcaatgg 960

gtgtgtaata?cgggcaacct?gaaccaacga?tttgggtttg?aagaagtcgc?gacgggagag 1020

tatttaatta?aatccaaacg?tagtcagctg?tgcttagagc?tcaagcccgg?tagtaatcaa 1080

tggcaggacg?gtgcgatcat?tcagcaatat?gtgtgtaatt?cagctgaaga?gaatcaacgt 1140

tggacgctat?ttgataaagg?cagccagacg?tttgagattc?gaagcaaggc?aacaggtaag 1200

tgtttagagc?ttgctgacaa?tcagggaagc?aacggcggga?cagtgcagca?atggtcatgt 1260

gatggtggta?ataaccagcg?attgaaattt?atacaactcg?agtgaatcga?attcctgcag 1320

cccgcatcgc?aggatgctgc?tggctaccct?gtggaacacc?tacatctgta?ttaacgaagc 1380

gctggcattg?accctgagtg?atttttctct?ggtcccgccg?catccatacc?gccagttgtt 1440

taccctcaca?acgttccagt?aaccgggcat?gttcatcatc?agtaacccgt?atcgtgagca 1500

tcctctctcg?tttcatcggt?atcattaccc?ccatgaacag?aaatccccct?tacacggagg 1560

catcagtgac?caaacaggaa?aaaaccgccc?ttaacatggc?ccgctttatc?agaagccaga 1620

cattaacgct?tctggagaaa?ctcaacgagc?tggacgcgga?tgaacaggca?gacatctgtg 1680

aatcgcttca?cgaccacgct?gatgagcttt?accgcagctg?cctcgcgcgt?ttcggtgatg 1740

acggtgaaaa?cctctgacac?atgcagctcc?cggagacggt?cacagcttgt?ctgtaagcgg 1800

atgccgggag?cagacaagcc?cgtcagggcg?cgtcagcggg?tgttggcggg?tgtcggggcg 1860

cagccatgac?ccagtcacgt?agcgatagcg?gagtgtatac?tggcttaact?atgcggcatc 1920

agagcagatt?gtactgagag?tgcaccatat?gcggtgtgaa?ataccgcaca?gatgcgtaag 1980

gagaaaatac?cgcatcaggc?gctcttccgc?ttcctcgctc?actgactcgc?tgcgctcggt 2040

cgttcggctg?cggcgagcgg?tatcagctca?ctcaaaggcg?gtaatacggt?tatccacaga 2100

atcaggggat?aacgcaggaa?agaacatgtg?agcaaaaggc?cagcaaaagg?ccaggaaccg 2160

taaaaaggcc?gcgttgctgg?cgtttttcca?taggctccgc?ccccctgacg?agcatcacaa 2220

aaatcgacgc?tcaagtcaga?ggtggcgaaa?cccgacagga?ctataaagat?accaggcgtt 2280

tccccctgga?agctccctcg?tgcgctctcc?tgttccgacc?ctgccgctta?ccggatacct 2340

gtccgccttt?ctcccttcgg?gaagcgtggc?gctttctcat?agctcacgct?gtaggtatct 2400

cagttcggtg?taggtcgttc?gctccaagct?gggctgtgtg?cacgaacccc?ccgttcagcc 2460

cgaccgctgc?gccttatccg?gtaactatcg?tcttgagtcc?aacccggtaa?gacacgactt 2520

atcgccactg?gcagcagcca?ctggtaacag?gattagcaga?gcgaggtatg?taggcggtgc 2580

tacagagttc?ttgaagtggt?ggcctaacta?cggctacact?agaaggacag?tatttggtat 2640

ctgcgctctg?ctgaagccag?ttaccttcgg?aaaaagagtt?ggtagctctt?gatccggcaa 2700

acaaaccacc?gctggtagcg?gtggtttttt?tgtttgcaag?cagcagatta?cgcgcagaaa 2760

aaaaggatct?caagaagatc?ctttgatctt?ttctacgggg?tctgacgctc?agtggaacga 2820

aaactcacgt?taagggattt?tggtcatgag?attatcaaaa?aggatcttca?cctagatcct 2880

tttaaattaa?aaatgaagtt?ttaaatcaat?ctaaagtata?tatgagtaaa?cttggtctga 2940

cagttaccaa?tgcttaatca?gtgaggcacc?tatctcagcg?atctgtctat?ttcgttcatc 3000

catagttgcc?tgactccccg?tcgtgtagat?aactacgata?cgggagggct?taccatctgg 3060

ccccagtgct?gcaatgatac?cgcgagaccc?acgctcaccg?gctccagatt?tatcagcaat 3120

aaaccagcca?gccggaaggg?ccgagcgcag?aagtggtcct?gcaactttat?ccgcctccat 3180

ccagtctatt?aattgttgcc?gggaagctag?agtaagtagt?tcgccagtta?atagtttgcg 3240

caacgttgtt?gccattgctg?caggcatcgt?ggtgtcacgc?tcgtcgtttg?gtatggcttc 3300

attcagctcc?ggttcccaac?gatcaaggcg?agttacatga?tcccccatgt?tgtgcaaaaa 3360

agcggttagc?tccttcggtc?ctccgatcgt?tgtcagaagt?aagttggccg?cagtgttatc 3420

actcatggtt?atggcagcac?tgcataattc?tcttactgtc?atgccatccg?taagatgctt 3480

ttctgtgact?ggtgagtttg?gttaattggt?taagactaat?ctttttgagt?agctcgtagt 3540

ttactttgac?gttaaataag?tatagtccta?atagttatgg?tataaaaact?ttttcggcaa 3600

agacattact?tcctcttttg?agtggctccg?tcaaggtatc?ctaccgttct?aggaccatag 3660

ccagacgcta?aggctgagca?ggttgtagtt?atgttggata?attaaagggg?agcagttttt 3720

attccaatag?ttcactcttt?agtggtactc?actgctgact?taggccactc?ttaccgtttt 3780

cgaatacgta?aagaaaggtc?tgaacaagtt?gtccggtcgg?taatgcgagc?agtagtttta 3840

gtgagcgtag?ttggtttggc?aataagtaag?cactaacgcg?gactcgctct?gctttatgcg 3900

ctagcgacaa?ttttcctgtt?aatgtttgtc?cttagcttac?gttggccgcg?tccttgtgac 3960

ggtcgcgtag?ttgttataaa?agtggactta?gtcctataag?aagattatgg?accttacgac 4020

aaaagggccc?ctagcgtcac?cactcattgg?tacgtagtag?tcctcatgcc?tattttacga 4080

actaccagcc?ttctccgtat?ttaaggcagt?cggtcaaatc?agactggtag?agtagacatt 4140

gtagtaaccg?ttgcgatgga?aacggtacaa?agtctttgtt?gagaccgcgt?agcccgaagg 4200

gtatgttagc?tatctaacag?cgtggactaa?cgggctgtaa?tagcgctcgg?gtaaatatgg 4260

gtatatttag?tcgtaggtac?aaccttaaat?tagcgccgga?gctcgttctg?caaagggcaa 4320

cttataccga?gtattgtggg?gaacataatg?acaaatacat?tcgtctgtca?aaataacaag 4380

tactactata?taaaaataga?acacgttaca?ttgtagtctc?taaaactctg?tgttgcaccg 4440

aaacaactta?tttagcttga?aaacgactca?acttcctagt?ctagtgcgta?gaagggctgt 4500

tgcgtctggc?aaggcaccgt?ttcgttttca?agttttagtg?gttgaccagg?tggatgttgt 4560

ttcgagagta?gttggca 4577

Claims (6)

1. the carrier of a catching secretion sequence, it is characterized in that: the carrier of catching secretion sequence is a base sequence among the sequence table SEQ ID No:2, and it contains the agarase gene agaV of the number of writing to sequence, and foreign gene inserts site and selection markers.

2. construction process by the described secretion sequence capturing carrier of claim 1 is characterized in that:

1) design plasmid pBK:

A. be template with plasmid pACYC177,, use restriction enzyme BamHI and HincII double digestion behind the PCR product purification with the kanamycin gene primer that contains BamHI site and HincII site respectively, pcr amplification kanamycin gene;

Wherein: the PCR condition is: 94 ℃ of pre-sex change template DNAs of 60s, 94 ℃ of 40s then, 45 ℃ of 60s, 72 ℃ of 60s change 94 ℃ of 40s into after 5 circulations, 58 ℃ of 60s, 72 ℃ of 60s, after 25 circulations again at 72 ℃ of extension 10min;

B. plasmid pBR322 is cut with BamHI and ScaI enzyme, electrophoresis reclaims the 3471bpDNA fragment, cut the PCR product of handling and be connected obtaining enzyme in this fragment and a step, connect liquid transformed into escherichia coli DH5 α, incubated overnight in containing kantlex LB substratum, filter out the transformant of anti-kantlex, from transformant, extract recombinant plasmid, be pBK;

2) design plasmid pUALS:

A. the operon of agaV is cloned into plasmid pUC19, constitute recombinant plasmid pBUA1, be template then with plasmid pBUA1, primer with containing BamHI site and XhoI site respectively carries out pcr amplification, obtains the PCR product of the agaV of the number of writing to sequence, with the PCR product purification, then be connected in room temperature, connect mixed solution transformed into escherichia coli DH5 α, on the LB solid medium that contains 100ug/ml Ap, 40ug/ml Xgal and 24ug/ml IPTG, cultivate the white transformant of screening with carrier pBS-T;

Wherein: the PCR condition: 94 ℃ of pre-sex change template DNAs of 60s, 94 ℃ of 40s then, 46 ℃ of 60s, 72 ℃ of 60s change 94 ℃ of 40s into after 5 circulations, 59 ℃ of 60s, 72 ℃ of 60s, after 25 circulations again at 72 ℃ of extension 10min;

B. from the white transformant of picking, extract recombinant plasmid, be pUALS;

3) design secretion sequence capturing carrier:

A. the plasmid pBK that obtains in the step 1) is cut with restriction enzyme BamHI and BsaBI enzyme, obtain the 3.2kbDNA fragment; With step 2) in plasmid pUALS with restriction enzyme BamHI and Sma I double digestion, obtain the 1.3kb dna fragmentation, it is connected with preceding 3.2kb dna fragmentation, connects and containing the transformant that screens anti-kantlex on the LB solid medium of Kn after liquid is transformed into bacillus coli DH 5 alpha;

B. from the transformant of anti-kantlex, extract recombinant plasmid, be secretion sequence capturing carrier pBU.

3. by the described secretion sequence capturing carrier of claim 2 construction process, it is characterized in that: the primer among the described step 1) a is KnF25 '-AT GGATCCACGGTTGATGAGAGC-3 ', KnR35 '-GCC GTCGACCAATTAACCAATTC-3 '.

4. by the described secretion sequence capturing carrier of claim 2 construction process, it is characterized in that: described step 2) primer among a is UAF55 '-AT GGATCCGAAGACTGGCGAGAAATC-3 ' UAR35 '-CA CTCGAGTTGTATAAATTTCAATCGC-3 '.

5. by the described secretion sequence capturing carrier of claim 2 construction process, it is characterized in that: after described step 2) obtaining the PCR product purification of agaV of the number of writing to sequence, be connected 2-4 hour in room temperature with carrier pBS-T.

6. application by the described secretion sequence capturing carrier of claim 1, it is characterized in that: described carrier can be caught secreted protein gene in the prokaryotic organism.

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