CN101456905B - Bacteria 5 type secretory protein and construction method and application thereof - Google Patents
Bacteria 5 type secretory protein and construction method and application thereof Download PDFInfo
- Publication number
- CN101456905B CN101456905B CN 200810237788 CN200810237788A CN101456905B CN 101456905 B CN101456905 B CN 101456905B CN 200810237788 CN200810237788 CN 200810237788 CN 200810237788 A CN200810237788 A CN 200810237788A CN 101456905 B CN101456905 B CN 101456905B
- Authority
- CN
- China
- Prior art keywords
- plasmid
- secretory protein
- alpha
- bacteria
- bacillus coli
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Abstract
The invention relates to the field of molecular biology, genetic engineering and immunology, in particular to a bacillus type V secretory protein and the application of the self transportation and surface display functions of the protein in the vaccine antigen expression and transfer. The type V secretory protein has a basic group sequence as shown in the sequence list SEQ ID No.1. The application method comprises the following steps: taking pseudomonas fluorescens TSS1 as a template, obtaining the type V secretory protein through PCR, and establishing plasmid pAT2 through the type V secretory protein; and taking Edwardsiella tarda TX1 as a template, obtaining the antigen protein gene through PCR, and connecting the antigen protein gene with the plasmid pAT2 so as to obtain surface display type pAT18 which is turned into Escherichia coli to obtain the bacillus surface display type protective vaccine capable of being directly inoculated for immunity.
Description
Technical field
The present invention relates to molecular biology, genetically engineered and field of immunology, specifically a kind of Pseudomonas fluorescens 5 type secretory protein and construction process and application.
Background technology
The prokaryotic organism secretory protein can according to its in cell K+transport and be divided into several large classes.Wherein, 5 type secretory protein has very unique character.Be different from other secretory protein, 5 type secretory protein self has completely transport function, do not rely on any other oroteins movement system of cell, thereby this class secretory protein also is called self-transport protein.On functional structure, 5 type secretory protein has a secreting signal peptide that is positioned at the N end usually, " passenger " albumen territory that betransported that is positioned at front end, and the transport function territory of a C end.N end signal peptide instructs protein to enter matrix, and the transport function territory forms a transport passage at cytolemma subsequently, and " passenger " albumen that betransported is transported to outside the born of the same parents by this passage.Because each functional domain structure of this proteinoid is relatively independent, thereby these functional domains can be replaced by different heterologous proteins, " passenger " albumen that for example betransported can be replaced by some suitable heterologous protein, and this replacement does not affect the effect in N end signal peptide and C end transport function territory.
Summary of the invention
The object of the invention is to provide a kind of Pseudomonas fluorescens 5 type secretory protein and construction process and application.
For achieving the above object, the technical solution used in the present invention is:
Bacteria 5 type secretory protein: formed by the base sequence among the sequence table SEQ ID No.1.
The construction process of bacteria 5 type secretory protein:
1) plasmid pAT1 makes up: take Pseudomonas fluorescens TSS1 as template, carry out pcr amplification with primer AF11 and AR9, product is connected with carrier pBS-T, and connecting fluid transforms bacillus coli DH 5 alpha, gets plasmid pBSA1; To be connected with oligonucleotide L11 with the plasmid pBR322 that the ScaI enzyme is cut, connecting fluid transforms bacillus coli DH 5 alpha, gets plasmid p329; To be connected with the plasmid pBSA1 that cuts with the SwaI/SmaI enzyme with the plasmid p329 that the EcoRV enzyme is cut, connecting fluid transforms bacillus coli DH 5 alpha, gets plasmid pAT1; Described oligonucleotide L11 sequence is 5 '-AAATCCCGGTCTAGAATTT-3 ';
2) structure of plasmid pAT2: take Pseudomonas fluorescens TSS1 as template, carry out pcr amplification with primer AF12 and AR12, product is connected with carrier pBS-T, connecting fluid transforms bacillus coli DH 5 alpha, get plasmid pBSA2, the 2kb fragment that will reclaim with the plasmid pBSA2 that the ScaI enzyme is cut is connected with the 5.1kb fragment that the plasmid pAT1 that cuts with the EcoRV enzyme reclaims, and connecting fluid is transformed into bacillus coli DH 5 alpha, gets plasmid pAT2;
3) structure of plasmid pAT18: take Edwardsiella tarda TX1 as template, carry out pcr amplification with primer ETF6 and ETR1, product is connected with the plasmid pAT2 that the ScaI enzyme is cut, connecting fluid transforms bacillus coli DH 5 alpha, gets the bacteria 5 type secretory protein that plasmid pAT18 has the base sequence among the sequence table SEQ ID No.1.
Described step 1) with ScaI digested plasmid pBR322, reclaim the 4.3kb fragment in, then it is connected 2-4 hour with the T4DNA ligase enzyme in room temperature with oligonucleotide L11, connecting fluid is transformed into bacillus coli DH 5 alpha.Described step 1) with EcoRV digested plasmid p329, reclaims the 4.4kb fragment in; It is connected with recovery 650bp fragment with SwaI/SmaI digested plasmid pBSA1, connects 2-4 hour with the T4DNA ligase enzyme in room temperature, connecting fluid is transformed into bacillus coli DH 5 alpha.Described step 3) the PCR product is connected 2-4 hour with the 7.1kb fragment that ScaI digested plasmid pAT2 reclaims in room temperature in, connects mixed solution and transforms bacillus coli DH 5 alpha.Described being transformed into behind the bacillus coli DH 5 alpha all cultivated 18-24 hour at the LB solid medium that contains 100ug/ml peace card penicillin, 40ug/ml Xgal and 24ug/ml isopropyl-β-D-thiogalactoside(IPTG), screened white transformant plasmid.With described bacteria 5 type secretory protein incubated overnight in the LB of the Ap that contains 100ug/ml liquid nutrient medium with the base sequence among the sequence table SEQ ID No.1, nutrient solution after getting 1ml and spending the night adds it in fresh LB liquid nutrient medium of 100ml, in 37 ℃ of lower wave and culture to OD
600Be 0.8, then centrifugal, collect bacterium liquid, namely get the vaccine antigen of expressing the bacteria 5 type secretory protein with the base sequence among the sequence table SEQ ID No.1, it has immunologic function.Described with the 160-200rpm wave and culture, centrifugal condition is 5000g, 4 ℃, and 10min.
The present invention has following advantage:
1. applicability is wide.5 type secretory protein tool of the present invention oneself transportation function does not rely on the protein transport system of host cell, and it is outer and be showed in bacterium surface that heterologous antigen albumen that can it is entrained successfully is transported to born of the same parents.Thereby can oneself's secretion in multiple Gram-negative bacteria.In addition, 5 type secretory protein of the present invention is low for structure and the size requirements of heterologous protein, can tolerate the insertion of large-scale heterologous protein, so can be used as the transport agent of dissimilar heterologous proteins.
2. high protection ratio.Pfa surface display vaccine of the present invention reaches 80% to the immunoprotection efficient of Edwardsiella tarda.
3. time saving and energy saving, simple.Vaccine antigen preparation process of the present invention is simple, need not proteins extraction, purifying etc.
Description of drawings
Fig. 1 is that (Fig. 1 wherein: whether Western immunoblot experiment detects the DH5 α that contains pAT18 can be shown to epicyte with vaccine to vaccine antigen bacterium surface displaying figure.Swimming lane 1: molecular weight marker; Swimming lane 2,3,4 is respectively epicyte albumen, intracellular protein, matrix albumen).
Embodiment
The invention will be further described below in conjunction with embodiment.Embodiment is intended to the description of giving an example to the present invention, but not limits the invention in any form.
Involved routinely experimental technique all adopts following method in embodiments of the present invention:
1. plasmid extraction, DNA (PCR) product purification, dna fragmentation reclaim from gel, the bacterial genomes DNA extraction is all used the corresponding reagent box of " TIANGEN Biotech (Beijing) Co., Ltd. ".
2. plasmid, the conversion of DNA connecting fluid enter intestinal bacteria and all use Hanahan 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.
4. all antibiotic working concentrations are as follows: peace card penicillin (Ap), 100ug/ml; Kantlex (Kn), 50ug/ml; Paraxin (Cm), 30ug/ml; Tsiklomitsin (Tc), 15ug/ml.
Embodiment 1
5 type secretory protein of the present invention is comprised of the pfa1 base sequence among the sequence table SEQ ID No.1.
5 type secretory protein construction process of the present invention:
1) structure of plasmid pAT1: take Pseudomonas fluorescens TSS1 as template, use following primer PCR: AF11 (5 '-CGCGGCGTTAACATTTAAATTGATGACGCACATATCCA-3 '), AR9 (5 '-CCCGGGATATCAGTACTATTGACCTGAGCTTCC-3 '), the PCR condition is: 94 ℃ of 60s denaturation template DNAs, then 94 ℃ of 40s, 51 ℃ of 60s, 72 ℃ of 60s, change 94 ℃ of 40s after 5 circulations into, 66 ℃ of 60s, 72 ℃ of 60s, after 25 circulations again at 72 ℃ of extension 10min.The PCR product with day root DNA product purification test kit purifying after with PCR cloning vector pBS-T (available from " day root biochemical technology company limited ", Beijing) connect 2-4 hour in room temperature, connect and cultivated 24-30 hour at the LB solid medium that contains 100ug/ml peace card penicillin (Ap), 40ug/ml Xgal (5-bromo-4-chloro-3-indoles-β-D-lactoside) and 24ug/ml isopropyl-β-D-thiogalactoside(IPTG) (IPTG) after mixed solution transforms bacillus coli DH 5 alpha, filter out white transformant, extract plasmid, be pBSA1.Plasmid pBR322 (is purchased from " Niu Yinglun Bioisystech Co., Ltd ", Beijing) cut with the ScaI enzyme, reclaim the 4.3kb fragment, with itself and oligonucleotide L11, wherein oligonucleotide L11 is 5 '-AAATCCCGGTCTAGAATTT-3 ', connects 2-4 hour with the T4DNA ligase enzyme in room temperature, connecting fluid was cultivated 24-30 hour at the LB solid medium that contains peace card penicillin after being transformed into bacillus coli DH 5 alpha, 1 transformant of picking extracts plasmid, with this plasmid called after p329.P329 is cut with the EcoRV enzyme, reclaim the 4.4kb fragment; Above-mentioned pBSA1 is cut with the SwaI/SmaI enzyme, reclaim the 650bp fragment, it is connected 2-4 hour with the T4DNA ligase enzyme in room temperature with aforementioned 4.4kb fragment, connecting fluid was cultivated 24-30 hour at the LB solid medium that contains peace card penicillin after being transformed into bacillus coli DH 5 alpha, 1 transformant of picking, extract plasmid, be pAT1.
Described LB solid medium moiety is by weight percentage: 1.0% peptone, 0.5% yeast powder, 1.0% sodium-chlor, 97.5% distilled water; Described Pseudomonas fluorescens TSS1 is stored in the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms, and deposit number is: CGMCCNo.2329.
2) structure of plasmid pAT2: take Pseudomonas fluorescens TSS1 as template, carry out pcr amplification with following primer, primer: AF12 (5 '-AGTACTACGCTGACCGGTGGC-3 '), AR12 (5 '-CAGTACTATTTAAATTAGAACAGGAAGGTAAACCCG-3 '), the PCR condition is: 94 ℃ of 60s denaturation template DNAs, then 94 ℃ of 40s, 57 ℃ of 60s, 72 ℃ of 60s, change 94 ℃ of 40s after 5 circulations into, 62 ℃ of 60s, 72 ℃ of 60s, after 25 circulations again at 72 ℃ of extension 10min.The PCR product is connected 2-4 hour with PCR cloning vector pBS-T in room temperature after using day root DNA product purification test kit purifying, connect and cultivated 24-30 hour at the LB solid medium that contains Ap, Xgal and IPTG (concentration is the same) after mixed solution transforms bacillus coli DH 5 alpha, filter out white transformant, extract plasmid, with its called after pBSA2.PBSA2 is cut with the ScaI enzyme, reclaim the 2kb fragment; With above-mentioned steps 1) plasmid pAT1 cut with the EcoRV enzyme, reclaim the 5.1kb fragment, it is connected 2-4 hour with the T4DNA ligase enzyme in room temperature with aforementioned 2kb fragment, connecting fluid was cultivated 24-30 hour at the LB solid medium that contains Kn after being transformed into bacillus coli DH 5 alpha, 1 transformant of picking, extract plasmid, be plasmid pAT2.
3) structure of plasmid pAT18: take Edwardsiella tarda TX1 as template, usefulness Pfu enzyme and following primer PCR amplification protective antigen gene: ETF6 (5 '-
CTCGAGGGAGGAGGAGGAGGAGGAGGATGCGTCGCCGCCGT-3 ', the line base is the XhoI site), ETR1 (5 '-
CTCGAGCCCGGGCTTCAGCAGCGAGAACGCG-3 ', the line base is the XhoI site), the PCR condition is: 94 ℃ of 60s denaturation template DNAs, then 94 ℃ of 40s, 58 ℃ of 60s, 72 ℃ of 60s, change 94 ℃ of 40s after 5 circulations into, 72 ℃ of 100s, after 25 circulations again at 72 ℃ of extension 10min.A PCR product day root DNA product purification test kit purifying.With above-mentioned steps 2) plasmid pAT2 cut with the ScaI enzyme, reclaim the 7.1kb fragment, it is connected 2-4 hour with the PCR product of aforementioned purifying in room temperature, connect and cultivated 24-30 hour at the LB solid medium that contains Ap after mixed solution transforms bacillus coli DH 5 alpha, 1 transformant of picking, extract plasmid, get the bacteria 5 type secretory protein that plasmid pAT18 has the base sequence among the sequence table SEQ IDNo.1.Wherein said Edwardsiella tarda TX1 is stored in the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms, and deposit number is: CGMCC No.2330.
ATGGACGTAGCAGGTAATGGCTTCATTGTGTCGCAACGCAATCGCACCCCCCGTTTCAAGACCACACCCCTCACACCCAT
AGCACTCGGCCTGGCCTTATGGCTGGGCCACGGTTCCGTGGCCAGGGCAGACGACAACCCTTACACCCCGCAGGTATTGG
AATCGGCGTTCAGGACAGCCGTCGCCTCATTCGGCCCTGAGACTGCCGTGTACAAAAACCTCAGGTTTGCCTACGCGGAT
ATTGTCGATCTTGCGGCCAAGGACTTCGCGGCCCAGTCCGGCAAGTTCGATTCGGCCCTCAAGCAAAACTATGAGCTGCA
ACCCGAGAACCTGACCATTGGCGCCATGCTCGGCGACACCCGTCGGCCACTGGACTACGCTTCGCGCCTGGATTACTACC
GCAGCCGGCTGTTCAGCAACAGCGGCCGCTACACCACCAATATCCTCGATTTCTCCAAGGCCATTATCGCCAACCTGCCG
GCCGCCAAGCCTTACACCTACGTCGAGCCGGGCGTGAGCAGCAACCTGAGCGGGCAGTTGAACGCAGGCCAGTCCTGGGC
TGGCGCAACCCGTGACTGGAGTGCCAACGCGCAAACCTGGAAGACCCCGGAAGCTCAGGTCAATCTCGAGGGAGGAGGAGGAGGAG
GAGGATGCGTCGCCGCCGTGATCGGCAGTGCGACCATGGCAACCCAGGCGGCCAGCGATCCCCGCAGCGTGGGTACCCAGGTCGACGA
TGGTACGCTGGAGGCCCGCATCTCCAACGCCCTGAGCAAAGACGCACAGCTGAAGAAAGAGGCGCGCGTCGTGGTAACCGCCTATCAG
GGGCAGGTGCTGCTGACCGGTCAGGCGCCAAGCCAGGCGCTGATTAGCCGCGCCAAGCAGATCGCGATGGGCGTTGAAGGCACCAAG
GCGGTCTATAATGAGATCCGTTTAGGCCAGCCGGTCAGCCTGGGCACTGCCTCGGCCGATGCCTGGATCACCACCAAGGTCCGCTCCCA
GCTGCTGGCCAGCGACCAGGTGAAATCCACCAACGTGAAGGTGACCACCGAAAATGGCGAGGTCTTCCTGCTGGGGCTGGTGACGCCC
AAAGAGGGACAGGCCGCCGCGCAAACGGCCAGTAAAGTCAGCGGGGTAAAACATGTCACTACCGCGTTCTCGCTGCTGAAGCTCGAG
CCCGGGACGCTGACCGGTGGCCGCACCTACGACAACCTGCAACCGGTGCATGATGCGCCGCCGGGTACGCCGCAAGTGCCAGGTGTGG
TCAGTGGTTGGGGCTTGCCCAACCTGCAAAAAGCCATGCAAGGGCCGGGGCAGTTCTTCGGTGCGGTGGCAGTGGCTTTGCCCAGCGG
TACCCGCGATATCTGGGCTAACCCGATTTCCGATGAAGCCATTCGCGCCCGTCGCGTAGAGGACGCTGCCGAACAGGCGACCTGGGCCG
CCACTAAAGCGCAAAAAGGCTGGCTCAATGGCCTGCCCGCCAATGCCTCGGCCGATGATCAGTTCGAATACGACATCGGTCATGCCCGG
GAGCAGGCAACACTCACCCGCGGCCAGGACGCGCTCACGGGCAGTACCTACGTCGGTAGCCTGGTCAAGTCCGGGGATGGCGAGCTGG
TGCTGGAAGGCCAGAACAGCTATTCGGGCAGCACTTGGGTACGCGGTGGCAAATTGTCGGTGGACGGCGCGTTGACCTCTGCCGTGAC
GGTAGATGGCAGCGCCGTGGGCATGCGCAATGCCGATAACGGCGTGATGACCACACTGGGCGGCACCCTGGCCGGCAACGGCACGGTG
GGTGCCTTGACCGTCAACAACGGCGGGCGAGTGGCCCCGGGGCATTCGA
TTGGCACGCTGCGCACCGGCGATGTCATGTTCAATCCGGGTTCGGTGTATGCCGTCGAAGTCGGCGCCAATGGTCAGAGC
GACCAGCTCCAGAGCAATGGCGTGGCGACTCTCAACGGTGGTGTGGTGAACGTGTCCCTGGAGAACAGCCCCAATCTGTT
GACCGCCACCGAGGCGCGCAGCTTGCTGGGCCAGCAGTTCACTATCCTCAGCGCCAGCCAAGGCATCCAGGGGCAGTTTG
CGGCGTCCGCCCCCAACTACCTGTTCATTGGCACTGCGCTCAACTATCAACCGAACCAGTTGACCCTGGCGATAGCCCGC
AACCAGACCACCTCCGCCAGCGTCGCGCAAACCCGCAATGAGCGGTCGGTGGCGACGGCAGCCGAGACATTGGGCGCTGG
CAGCCCGGTCTACGAAAGCCTGCTGGCGTCGGATTCCGCCGCTCAGGCGCGGGAAGGGTTCAGGCAACTGTCGGGGCAAC
TGCATTCGGATGTGGCGGCGGCGCAAATGGCCGACAGTCGCTACCTGCGTGAAGCGGTCAACGCTCGCCTGCAACAGGCG
CAGGCACTGGACTCCAGCGCGCAGATCGACAGCCGTGACAACGGCGGCTGGGTACAGCTGCTTGGTGGACGCAACAACGT
CAGTGGTGACAACAACGCCAGCGGCTACTCCTCGTCCACCAGCGGCGTACTGCTGGGCCTGGACTCCGAGGTGAACGACG
GCTGGCGCGTGGGCGCGGCGACCGGTTATACCCAAAGCCACCTCAACGGCCAGTCGGCGTCGGCGGACAGCGACAACTAT
CACCTGTCGGTCTATGGCGGCAAACGCTTCGAGGCGATTGCCCTGCGCCTGGGCGGTGCCAGCACCTGGCACCGTCTGGA
CACTTCGCGACGGGTGGCCTATGCCAATCAGTCGGACCATGCCAAGGCCGACTACAACGCGCGTACCGACCAAGTGTTTG
CCGAG?ATCGGTTACACCCAGTGGACCGTGTTTGAACCCTTCGCCAACCTCACGTACCTGAACTATCAAAGCGACTCGTTC
AAGGAAAAAGGCGGTGCCGCAGCCTTGCATGCCAGCCAGCAAAGCCAGGACGCGACACTCTCCACCCTGGGCGTGCGTGG
CCATACCCAGCTGCCGCTCACGTCCACCTCGGCGGTGACCCTGCGCGGTGAGCTGGGTTGGGAGCACCAGTTTGGTGATA
CCGATCGTGAAGCTTCTCTGAAGTTTGCCGGCAGTGACACGGCCTTCGCCGTCAACAGCGTGCCTGTGGCCAGGGATGGC
GCGGTGATCAAGGCCAGTGCACAAATGGCGCTGACCAAGGACACCCTGGTGTCATTGAACTACAGTGGCTTGCTGTCCAA
CCGCGGTAACAACAACGGGATCAATGCCGGGTTTACCTTCCTGTTCTAA
(a) sequence signature:
● length: 3249bp
● type: base sequence
● chain: strand
● topological framework: linearity
(b) molecule type: double-stranded DNA
(c) suppose: no
(d) antisense: no
(e) initial source: Pseudomonas fluorescens TSS1
(f) specificity title: pfa
N end: front 624 bases; Middle portion: middle 555 bases (are base 625-1179; );
C end: last 2070 bases (being base 1180-3249).
Embodiment 2
Surface display type vaccine preparation: will contain bacillus coli DH 5 alpha incubated overnight in containing the LB substratum of Ap of plasmid pAT18 that above-described embodiment 1 gained has the bacteria 5 type secretory protein of the base sequence among the sequence table SEQ ID No.1; Nutrient solution after getting 1ml and spending the night adds it in fresh LB liquid nutrient medium of 100ml, is 0.9 in 37 ℃ of lower rotating speed 160r-200pm wave and culture to OD600.Then with inoculum centrifugal (5000g, 4 ℃, 10min), collect bacterium liquid, namely get surface display type vaccine.This vaccine is owing to be to be inserted in the middle of the 5 type secretory protein Pfa, thereby carried secretion to the extracellular by Pfa, is overlying on cell surface with the form of protein, so be called surface display type antigen.The Westernimmunoblot experimental result shows that the bacillus coli DH 5 alpha that contains pAT18 can be shown to cell surface (being adventitia) (referring to Fig. 1) with vaccine by Pfa really.
The immunity of vaccine is used:
Step 1) immunization of vaccine.With the collected thalline of above-mentioned surface display type vaccine be suspended among the PBS to final concentration be 5 * 10
8Cfu/ml is vaccine and prepares liquid.60 lefteye flounders (every heavily about 14g) are divided into 2 groups, 30 every group at random.With these 2 groups difference called after A and B group.With every fish of A group respectively the above-mentioned vaccine of abdominal injection 100ul prepare liquid.Every fish difference abdominal injection 100ul PBS with the B group.
Wherein the PBS moiety is: 0.8g NaCl, 0.02g KCl, 0.358g Na
2HPO
4.12H
2O, 0.024g NaH
2PO
4
Step 2) preparation of Edwardsiella tarda suspension.In the LB substratum, cultivate respectively Edwardsiella tarda TX1 to OD
600Be 0.5, centrifugal (5000g, 4 ℃) 10min then.Collect thalline, with its be suspended among the PBS to final concentration be 1 * 10
7Cfu/ml is the Edwardsiella tarda suspension.
Step 3) immunoprotective effec take 5 type secretory protein Pfa1 as the surface display type vaccine on basis detects.In step 1) after for the first time immunization the 32nd day, with above-mentioned steps 2) Edwardsiella tarda suspension abdominal injection step 1) A and B group fish, the injection volume of every fish is 100ul.In afterwards 21 days, observe and record the death condition of respectively organizing fish every day.After 21 days, statistics is respectively organized total mortality of fish: A group, 5; The B group, 24.Utilize following formula to calculate premunition protection efficient (RPS):
RPS=100 * (the total dead per-cent of the total dead per-cent of 1-immune group fish/control group fish)
Drawing thus vaccine is 80% for the immunoprotection efficient of Edwardsiella tarda, infects so it can protect lefteye flounder to resist Edwardsiella tarda effectively.
Claims (6)
1. bacteria 5 type secretory protein, it is characterized in that: the bacteria 5 type secretory protein base sequence is shown in the sequence table SEQ ID No.1.
2. construction process by bacteria 5 type secretory protein claimed in claim 1 is characterized in that:
1) plasmid pAT1 makes up: take Pseudomonas fluorescens TSS1 as template, carry out pcr amplification with primer AF11 and AR9, product is connected with carrier pBS-T, and connecting fluid transforms bacillus coli DH 5 alpha, gets plasmid pBSA1; To be connected with oligonucleotide L11 with the plasmid pBR322 that the ScaI enzyme is cut, connecting fluid transforms bacillus coli DH 5 alpha, gets plasmid p329; To be connected with the plasmid pBSA1 that cuts with the SwaI/SmaI enzyme with the plasmid p329 that the EcoRV enzyme is cut, connecting fluid transforms bacillus coli DH 5 alpha, gets plasmid pAT1; Described oligonucleotide L11 sequence is 5 '-AAATCCCGGTCTAGAATTT-3 ';
2) structure of plasmid pAT2: take Pseudomonas fluorescens TSS1 as template, carry out pcr amplification with primer AF12 and AR12, product is connected with carrier pBS-T, connecting fluid transforms bacillus coli DH 5 alpha, get plasmid pBSA2, the 2kb fragment that will reclaim with the plasmid pBSA2 that the ScaI enzyme is cut is connected with the 5.1kb fragment that the plasmid pAT1 that cuts with the EcoRV enzyme reclaims, and connecting fluid is transformed into bacillus coli DH 5 alpha, gets plasmid pAT2;
3) structure of plasmid pAT18: take Edwardsiella tarda TX1 as template, carry out pcr amplification with primer ETF6 and ETR1, product is connected with the plasmid pAT2 that the ScaI enzyme is cut, connecting fluid transforms bacillus coli DH 5 alpha, gets the bacteria 5 type secretory protein that plasmid pAT18 has the base sequence among the sequence table SEQ ID No.1.
3. press the construction process of bacteria 5 type secretory protein claimed in claim 2, it is characterized in that: use ScaI digested plasmid pBR322 described step 1), reclaim the 4.3kb fragment, then it is connected 2-4 hour with the T4DNA ligase enzyme in room temperature with oligonucleotide L11, connecting fluid is transformed into bacillus coli DH 5 alpha.
4. by the construction process of bacteria 5 type secretory protein claimed in claim 2, it is characterized in that: with EcoRV digested plasmid p329, reclaim the 4.4kb fragment described step 1); It is connected with recovery 650bp fragment with SwaI/SmaI digested plasmid pBSA1, connects 2-4 hour with the T4DNA ligase enzyme in room temperature, connecting fluid is transformed into bacillus coli DH 5 alpha.
5. by the construction process of bacteria 5 type secretory protein claimed in claim 2, it is characterized in that: the PCR product is connected 2-4 hour with the 7.1kb fragment that ScaI digested plasmid pAT2 reclaims in room temperature described step 3), connects mixed solution and transforms bacillus coli DH 5 alpha.
6. press the construction process of bacteria 5 type secretory protein claimed in claim 2, it is characterized in that: described being transformed into behind the bacillus coli DH 5 alpha all cultivated 18-24 hour at the LB solid medium that contains 100ug/ml peace card penicillin, 40ug/mlXgal and 24ug/ml isopropyl-β-D-thiogalactoside(IPTG), screened white transformant plasmid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810237788 CN101456905B (en) | 2008-12-05 | 2008-12-05 | Bacteria 5 type secretory protein and construction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810237788 CN101456905B (en) | 2008-12-05 | 2008-12-05 | Bacteria 5 type secretory protein and construction method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101456905A CN101456905A (en) | 2009-06-17 |
| CN101456905B true CN101456905B (en) | 2013-01-16 |
Family
ID=40768051
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200810237788 Expired - Fee Related CN101456905B (en) | 2008-12-05 | 2008-12-05 | Bacteria 5 type secretory protein and construction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101456905B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102614504B (en) * | 2012-03-13 | 2013-12-04 | 中国科学院海洋研究所 | Pseudomonas fluorescens recombinant protein vaccine and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1136280A (en) * | 1994-07-29 | 1996-11-20 | 味之素株式会社 | Anti-aids secretory recombinant BCG vaccine |
| CN1738901A (en) * | 2002-10-11 | 2006-02-22 | 伊姆维申股份有限公司 | Modular antigen transporter molecules (MAT molecules) for modulating immune reactions, associated constructs, methods and uses |
| CN101062410A (en) * | 2007-02-05 | 2007-10-31 | 中国人民解放军第三军医大学 | Genetic engineering vaccine of enterohemorrhagic escherichia coli 0157:H7 and the preparing method thereof |
-
2008
- 2008-12-05 CN CN 200810237788 patent/CN101456905B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1136280A (en) * | 1994-07-29 | 1996-11-20 | 味之素株式会社 | Anti-aids secretory recombinant BCG vaccine |
| CN1738901A (en) * | 2002-10-11 | 2006-02-22 | 伊姆维申股份有限公司 | Modular antigen transporter molecules (MAT molecules) for modulating immune reactions, associated constructs, methods and uses |
| CN101062410A (en) * | 2007-02-05 | 2007-10-31 | 中国人民解放军第三军医大学 | Genetic engineering vaccine of enterohemorrhagic escherichia coli 0157:H7 and the preparing method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 张斌等.结核分枝杆菌抗原ESAT26和CFP210及rCFP102ESAT6融合基因在大肠埃希菌中的表达及比较.《中华实验和临床感染病杂志》.2008,第2卷(第1期),33-43. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101456905A (en) | 2009-06-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hoshina et al. | Chlorella variabilis and Micractinium reisseri sp. nov.(Chlorellaceae, Trebouxiophyceae): Redescription of the endosymbiotic green algae of Paramecium bursaria (Peniculia, Oligohymenophorea) in the 120th year | |
| Golanowska et al. | Comparison of highly and weakly virulent Dickeya solani strains, with a view on the pangenome and panregulon of this species | |
| Phelan et al. | Diversity and bioactive potential of endospore‐forming bacteria cultured from the marine sponge Haliclona simulans | |
| Van Keulen et al. | Gas vesicles in actinomycetes: old buoys in novel habitats? | |
| CN103352015B (en) | HtpsA-gene-knock-out mutant strain of Streptococcus suis serotype 2 and application thereof | |
| CN107164395A (en) | A kind of molecular labeling of algal toxin degradation bacterium, algal toxin degradation bacterium and the preparation method of algal toxin degradation bacterium | |
| CN101848930A (en) | Plasmid-encoded neurotoxin genes in clostridium botulinum serotype a subtypes | |
| CN101456905B (en) | Bacteria 5 type secretory protein and construction method and application thereof | |
| CN104630184B (en) | A kind of alkalescent xylanase and its encoding gene and application | |
| CN109593743A (en) | Novel C RISPR/ScCas12a albumen and preparation method thereof | |
| CN102719413A (en) | Novel tannase and application thereof | |
| CN101491671A (en) | Cross-protection vaccine and building method thereof | |
| CN101475944B (en) | Promoter replacement method for improving Bacillus amyloliquefaciens yield | |
| CN106467898B (en) | A kind of endogenetic fungal bacterial strain and its metabolite extracting method and purposes | |
| CN102614504A (en) | Pseudomonas fluorescens recombinant protein vaccine and preparation method thereof | |
| CN103215215A (en) | Escherichia coli expression strain with high conversion efficiency | |
| CN103910785B (en) | The outer membrane receptor of a kind of Pseudomonas fluorescens TonB dependent form and application thereof | |
| Orfei et al. | Race-specific genotypes of Pseudomonas syringae pv. tomato are defined by the presence of mobile DNA elements within the genome | |
| CN102028940B (en) | Edwardsiella tarda surface display type vaccine and preparation and application thereof | |
| CN104313042A (en) | Paenibacillus polymyxa neopullulanase gene as well as method for producing neopullulanase through cloning and expression and fermentation | |
| CN101519440A (en) | Intersecting protective vaccine antigen, preparation method and application thereof | |
| CN105821068A (en) | Recombinant vector, construction method of recombinant vector, engineering bacteria of fenpropathrin degrading esterase est3385, preparing method and application | |
| CN103041386A (en) | Application of vibrio anguillarum recombinant protein | |
| CN1482247A (en) | Non-antibiotics resistant shuttle plasmid expression carrier and its constructing method and uses | |
| CN102373202B (en) | Biphenyl degradation related gene operon derived from fluorescent pseudomonas and expression vector and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130116 Termination date: 20151205 |
|
| EXPY | Termination of patent right or utility model |