CN104800839B - The application of Pseudomonas fluorescens iron-regulatory protein - Google Patents
The application of Pseudomonas fluorescens iron-regulatory protein Download PDFInfo
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- CN104800839B CN104800839B CN201510010428.9A CN201510010428A CN104800839B CN 104800839 B CN104800839 B CN 104800839B CN 201510010428 A CN201510010428 A CN 201510010428A CN 104800839 B CN104800839 B CN 104800839B
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- iron
- pseudomonas fluorescens
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Abstract
The present invention relates to the application of field of molecular vaccinology, specifically Pseudomonas fluorescens iron-regulatory protein.Iron-regulatory protein is used for the immune vaccine for preparing Pseudomonas fluorescens.The iron-regulatory protein of the present invention can effectively protect lefteye flounder to resist Pseudomonas fluorescens to infect as vaccine.
Description
Technical field
The present invention relates to the application of field of molecular vaccinology, specifically Pseudomonas fluorescens iron-regulatory protein.
Background technology
Pseudomonas fluorescens is a kind of gramnegative bacterium, is distributed widely in various environment.Pseudomonas fluorescens is weight
The aquatic animal pathogenic bacteria wanted, aquatic vertebrate and invertebrate can be infected, the former includes a variety of culturing economic fish.
For fish, Pseudomonas fluorescens infection host scope is wide, both including freshwater fish or including seawater fish.At present to the bacterium
Caused disease there is no effective precautionary measures at home.Iron is nutritional ingredient necessary to a kind of bacteria live.In many bacteriums
In research show that iron regulates and controls the expression of a large amount of bacterioproteins, these albumen participate in various biological functions, including iron obtains and fortune
Defeated, metabolic response and pathogenic course etc..In some bacteriums, the iron-regulatory protein positioned at cell surface participates in bacterium infection mistake
Journey simultaneously has immanoprotection action.The research of current glimmering pseudomonad iron-regulatory protein is seldom, and its effect is also not very clear.
The content of the invention
Present invention aims at the application for providing Pseudomonas fluorescens iron-regulatory protein.
To achieve the above object, the technical solution adopted by the present invention is：
A kind of application of Pseudomonas fluorescens iron-regulatory protein, iron-regulatory protein are used to prepare the immune of Pseudomonas fluorescens
Vaccine.The iron-regulatory protein is plasmid pETTfeR or pETOprF.
Further, using Pseudomonas fluorescens TSS1 as template, F1/R1 or F2/R2 is used to enter performing PCR respectively for primer
Amplification, PCR primer are connected with carrier pET259 respectively, obtain plasmid pETTfeR or pETOprF, and gained plasmid converts greatly respectively
Enterobacteria BL21 (DE3) can express restructuring iron-regulatory protein.
The primer is F1：5’-GATATCATGAGCCCCAGCTTCAACGCCT-3’；R1：5’-
GATATCCGATGTAGTCACCGACGCGAA-3’；F2：5’-GATATCATGAAACTGAAAAACACCTTGG-3’；R2：5’-
GATATCCTGAGCGGTA GCTTCAACC-3’。
The invention has the advantages that：
1. notable protectiveness.The Pseudomonas fluorescens iron-regulatory protein of the present invention is exempted from as vaccine to Pseudomonas fluorescens
Epidemic disease protective efficacy is up to more than 50%.
2. the vaccine of the present invention need not be commercialized adjuvant.
Brief description of the drawings
Fig. 1 is the iron-regulatory protein (swimming lane 1) of purifying provided in an embodiment of the present invention.Swimming lane M, molecular weight standard.
Embodiment
With reference to embodiment, the invention will be further described.Embodiment is intended to carry out citing description to the present invention, and
It is non-to limit the invention in any form.Involved routinely experimental method is using such as in embodiments of the present invention
Lower method：
1. plasmid extraction, DNA (PCR) product purification all use the mutually examination of " TIANGEN Biotech (Beijing) Co., Ltd. "
Agent box.
2. the conversion of plasmid, DNA connection liquid all uses Hanahan methods (Sambrook and Russell into Escherichia coli:
Molecular Cloning:A Laboratory Mannual.Cold Spring Harbor Laboratory Press
2001)；
3. all restriction enzymes and ligase are purchased from " Beijing, knob Great Britain Bioisystech Co., Ltd ".
Embodiment 1
Pseudomonas fluorescens iron-regulatory protein TfeR and OprF expression are by iron conditional regulatory
Pseudomonas fluorescens TSS1 normal LB culture mediums (control group) or containing 600uM iron complexs 2,2 '-
Culture 2h is shaken in dipyridyl (being purchased from Sigma Co., USA) LB culture mediums, cultivation temperature is 28 DEG C, rotating speed 160rpm.
Then with 5000g, 4 DEG C of centrifugation 10min, thalline is collected.The thalline of collection is subjected to fluorescence quantitative PCR detection TfeR or OprF base
(specific method is shown in document for the expression of cause：Zhang SR,Zhang L,Sun L.Identification and analysis of
three virulence-associated TonB-dependent outer membrane receptors of
Pseudomonas fluorescens.Dis Aquat Org.2014；110:181-91.).As a result show, in iron complex 2,
In the presence of 2 '-dipyridyl, the expression quantity significantly (P of TfeR and OprF genes<0.01) up-regulation (TfeR that detects and
OprF mRNA amounts significantly increase) (being respectively 14 times and 30 times of control group), illustrate the two genes by iron conditional regulatory.
The LB constituents are by weight percentage：1.0% peptone, 0.5% dusty yeast, 1.0% sodium chloride,
97.5% distilled water；The bacterial strain TSS1 is stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center
CGMCC, deposit number are：CGMCC No.2329, Classification And Nomenclature are Pseudomonas fluorescens (Pseudomonas
Fluorescens) preservation date is on January 9th, 2008, and depositary institution address is Datun Road, Chaoyang District, Beijing City, Chinese science
Institute of microbiology of institute.
Embodiment 2
Recombinate Pseudomonas fluorescens iron-regulatory protein rTfeR and rOprF preparation
Step 1) rTfeR and rOprF expression vector pETTfeR and pETOprF structure：
Pseudomonas fluorescens TfeR and OprF sequence have been reported (GenBank access ion
Number.AFJ59752.1 and AFJ59305.1).PETTfeR structure is as follows：Using Pseudomonas fluorescens TSS1 as template, adopt
TfeR genes are expanded with primers F 1/R1.PCR conditions are：94 DEG C of 60s pre-degeneration template DNAs, then 94 DEG C of 40s, 60 DEG C of 60s,
72 DEG C of 60s, 5 circulate, then 94 DEG C of 40s, 66 DEG C of 60s, 72 DEG C of 60s, 30 circulations.The corresponding reagent of PCR primer Tiangeng
Box purifies.By expression vector pET259, (pET259 building process is referring to Hu YH, Zheng WW, Sun L.Identification
and molecular analysis of a ferritin subunit from red drum(Sciaenops
ocellatus).Fish Shellfish Immunol 2010；28:678-86) use restriction enzyme SwaI digestions after with it is upper
The PCR primer for stating purifying is connected with T4DNA ligases, and connection liquid is transformed into bacillus coli DH 5 alpha, containing kanamycins (50ug/
Ml cultivated on LB culture mediums) 18-24 hours, screening transformant extraction plasmid, as pETTfeR.
PETOprF structure is as follows：Using Pseudomonas fluorescens TSS1 as template, OprF bases are expanded using primers F 2/R2PCR
Cause.PCR conditions are：94 DEG C of 60s pre-degeneration template DNAs, then 94 DEG C of 40s, 56 DEG C of 60s, 72 DEG C of 60s, 5 circulations, then 94
DEG C 40s, 62 DEG C of 60s, 72 DEG C of 60s, 30 circulations.The corresponding reagent box purifying of PCR primer Tiangeng.By expression vector pET259
It is connected with after restriction enzyme SwaI digestions with the PCR primer of above-mentioned purifying with T4DNA ligases, connection liquid is transformed into large intestine
Bacillus DH5 α, cultivated on the LB culture mediums containing kanamycins (50ug/ml) 18-24 hours, screening transformant extraction plasmid,
As pETOprF.
The primer is F1：5’-GATATCATGAGCCCCAGCTTCAACGCCT-3’；R1：5’-
GATATCCGATGTAGTCACCGACGCGAA-3’；F2：5’-GATATCATGAAACTGAAAAACACCTTGG-3’；R2：5’-
GATATCCTGAGCGGTA GCTTCAACC-3’。
Step 2) restructuring Pseudomonas fluorescens iron-regulatory protein rTfeR and rOprF expression and purity：
RTfeR expression and purity is as follows：By above-mentioned steps 1) plasmid pETTfeR conventional method conversion large intestine bars
Bacterium BL21 (DE3) (is purchased from " Tiangeng biochemical technology Co., Ltd ", Beijing), is consolidated in the LB containing kanamycins (50ug/ml)
18-24 hours are cultivated on body culture medium, picking transformant, are named as BL21/pETTfeR.By BL21/pETTfeR in containing
Have in the LB fluid nutrient mediums of kanamycins (50ug/ml) and be incubated overnight；Nutrient solution after taking 1ml overnight, it is fresh to add 100ml
The LB fluid nutrient mediums containing kanamycins (50ug/ml) in, rotating speed 200rpm shakes culture to OD at 37 DEG C600For
0.6, add final concentration of 1mM IPTG, 37 DEG C are continued to shake culture 4-5h with rotating speed 160rpm, then with 5000g, 4 DEG C from
Heart 10min, bacterium solution is collected, add 5ml lysates, in room temperature in slowly shaking 1-2 hours on shaking table, until bacteria suspension becomes clarification
Untill.By bacterium solution with 10000g, 4 DEG C of centrifugation 30min, supernatant is reclaimed.By the His Trap HP Columns of the albumen in supernatant
(being purchased from GE Healthcare companies of the U.S.) recovery purifying, the albumen of purifying is through SDS-PAGE electrophoresis detections (under 8v/cm voltages
Electrophoresis 25-30min, electrophoresis 2-2.5h under subsequent 15v/cm voltages), determine its molecular size range, with rTfeR coincide (referring to
Figure 1A).
ROprF expression and purity is as follows：By above-mentioned steps 1) plasmid pETOprF conventional method conversion large intestine bars
Bacterium BL21 (DE3) (is purchased from " Tiangeng biochemical technology Co., Ltd ", Beijing), is consolidated in the LB containing kanamycins (50ug/ml)
18-24 hours are cultivated on body culture medium, picking transformant, are named as BL21/pETOprF.Subsequent all steps are same
RTfeR expression and purity.The albumen of purifying (is same as above) through SDS-PAGE electrophoresis detections, determines its molecular size range, with
ROprF coincide (referring to Figure 1B).
The lysate NaH containing 10mM2PO4, 10mM Tris and 8M urea, pH 8.0.
Embodiment 3
Recombinate applications of the Pseudomonas fluorescens iron-regulatory protein rTfeR and rOprF as vaccine
The preparation of step 1) adjuvant and vaccine mixed liquor.
The preparation of vehicle control liquid:By 5% (mass ratio) NaOH and 5% (mass ratio) Al2(SO4)3With 2：5 volume ratios are mixed
Close, mixture is centrifuged 5 minutes with 10,000g.Precipitation is suspended in PBS to 0.2mg/ml, as adjuvant.By PBS and assistant
Agent mixes in equal volume, as vehicle control liquid.
It is prepared by vaccine mixed liquor：The rTfeR or OprF that above-described embodiment 2 is purified are diluted to 200ug/ in PBS respectively
Ml, the albumen after dilution is mixed in equal volume with adjuvant, as rTfeR vaccines mixed liquor or rOprF vaccine mixed liquors.
The PBS constituents are by weight percentage：0.8%NaCl, 0.02%KCl, 0.358%
Na2HPO4.12H2O, 0.024%NaH2PO4, surplus is water.
The immune application of step 2) vaccine.120 lefteye flounders (every weighs about 18g) are randomly divided into 3 groups, every group 40.Will
This 3 groups are respectively designated as A, B and C groups.By every fish of A groups intraperitoneal injection 100ul above-mentioned steps 1) rTfeR vaccines mix
Liquid, 100ul above-mentioned steps 1 are injected intraperitoneally in every fish of B groups) rOprF vaccine mixed liquors, by every fish belly chamber note of C groups
Penetrate 100ul above-mentioned steps 1) vehicle control liquid.
The preparation of step 3) Pseudomonas bacteria suspension.Pseudomonas fluorescens TSS1 to OD is cultivated in LB culture mediums600For
1, it is then centrifuged for (5000g, 4 DEG C) 10min.Thalline is collected, is suspended in PBS to final concentration of 1x108cfu/ml。
Step 4) vaccine immunity protective effect detects.The 30th day after step 2) inoculation, with above-mentioned steps 3)
3 groups of fishes of step 2) are injected intraperitoneally in Pseudomonas bacteria suspension, and the injection volume of every fish is 100ul.In 20 days afterwards, often
It is observed and records the death condition of each group fish.After 20 days, total mortality of each group fish is counted：A groups, 20；B groups, 17；
C groups, 40.Relative immunity protective efficacy (RPS) is calculated using following equation：
RPS=100x (total Percent mortality of total Percent mortality/control group fish of 1- immune group fishes)
The relative immunity protective efficacy that rTfeR and rOprF is calculated according to this formula is respectively 50% and 58%.Therefore, weight
Group Pseudomonas fluorescens iron-regulatory protein rTfeR and rOprF effectively can protect lefteye flounder to resist Pseudomonas as vaccine
Bacterium is infected.
Claims (1)
- A kind of 1. application of Pseudomonas fluorescens iron-regulatory protein, it is characterised in that：Iron-regulatory protein is used to prepare the false list of fluorescence The immune vaccine of born of the same parents bacterium；Using Pseudomonas fluorescens TSS1 as template, F1/R1 or F2/R2 is used to enter performing PCR amplification, PCR primer point respectively for primer It is not connected with carrier pET259, obtains plasmid pETTfeR or pETOprF, gained plasmid converts e. coli bl21 DE3 i.e. respectively Restructuring iron-regulatory protein can be expressed；The primer is F1：5’-GATATCATGAGCCCCAGCTTCAACGCCT-3’；R1：5’- GATATCCGATGTAGTCACCGACGCGAA-3’；F2：5’-GATATCATGAAACTGAAAAACACCTTGG-3’；R2：5’- GATATCCTGAGCGGTAGCTTCAACC-3’。
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| 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 |
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