CN106191044A - A kind of gene engineering method improving lactein plnK yield - Google Patents
A kind of gene engineering method improving lactein plnK yield Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/335—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Lactobacillus (G)
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12N2800/101—Plasmid DNA for bacteria
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Abstract
The invention discloses a kind of gene engineering method improving lactein plnk yield.Gene order according to lactein plnk, design a pair specific primer, high efficient expression is selected to fuse the prokaryotic expression carrier PET 32a(+ of albumen), build pET 32a plnK recombiant plasmid, by the optimization of abduction delivering system is shown, abduction delivering temperature 16 43 DEG C, the abduction delivering time 8,16h, IPTG concentration 0.2 0.6 mmoL/L was optimal induction system.Bacteriostatic experiment after purification destination protein shows, in-vitro recombination expression lactein plnK still has higher fungistatic effect.
Description
Technical field
The invention belongs to veterinary drug and field of feed additive technology, be specifically related to improve the gene of lactein plnK yield
Engineering method.
Background technology
Antibiotic is widely applied in Animal husbandry production as poultry antibacterial medicines and growth promoter.But it is long-term
Use antibiotic, not only affect the prevention effect of Animal diseases；Also can develop immunity to drugs and remain, cause serious livestock products dirty
Dye, the humans and animals that even can induce having produces canceration and distortion, jeopardizes the health and safety of the mankind.Therefore, January 1 in 2006
Day rises, and European Union completely forbids food animal and uses antibiotic feed additive for promoting growth.Completely forbid antibiotic to move as food
Thing feed additive for promoting growth, increasingly causes the most attention in the whole world.But, European Union is owing to completely forbidding antibiotic feed
Additive also serves problem to animal productiong band, such as the increase of enterobacterial diseases.In order to reverse the decline of animal health, only
Therapeutic antibiotics can be used in a large number to treat.Simultaneously as the increase of intestinal bacteriosis too increases animal food
The chance polluted by intestinal cell is the most salmonella-polluted.To this end, develop one can improve livestock birds health, improve production performance and
The product of the energy substitute antibiotics of safety non-toxic has become a focus of Animal nutrition research.
Lactein is in lactic acid bacteria metabolic process, synthesizes and the class that is secreted in environment is to gram positive bacteria
Albumen that (antibacterial that especially affinity is nearer) and gram negative bacteria are inhibited or polypeptide.Due to lactein without
Toxic and side effects, noresidue, have no drug resistance, it is possible to suppress or kill some pathogenic bacterium, there is certain heat stability, the most passively
Object gastral Partial Protein enzymatic degradation, causes untoward reaction thus without accumulation in vivo.The treatment potentiality of bacteriocin are drawn
Play people's concern to this field.So far, only a kind of Lantibiotics Nisin is for food antiseptic, most
Under test, the wild strain of the mainly bacteriocinogeny of tracing it to its cause yields poorly, isolated and purified difficulty, distance industry metaplasia
Produce and also have very distance.Therefore, obtaining lactein by external technology is the important channel obtaining high yield lactein.
Lactein plnk is the one of lactein, has confirmed that it has the fungistatic effect of wide spectrum, yet with open country
Raw type lactic acid bacteria yields poorly and is difficult to and other Protein Separation, and hinders its industrialization and produce.Therefore, lactein is optimized
The production system of plnk has great importance for solving lactic acid bacteria industrialization production.
Summary of the invention
It is an object of the invention to provide a kind of gene engineering method improving lactein plnK yield.
For achieving the above object, the present invention adopts the following technical scheme that
A kind of gene engineering method improving lactein plnK yield, by building genes of interest recombinant expression plasmid, optimizes
Expression system, it is thus achieved that can the technique for gene engineering of great expression lactein plnk in vitro.
Method particularly includes:
1. a pair plnK specific primer of design, primer sequence is forward primer F:5 '-GCGGATCCATGAAAATTAAATTAAC
TGT-3’；Downstream trip primer R:5 '-GCGTCGACTCACTTATTATAATCCCTTG-3 '.
2. select that there is the excellent carrier PET32a expressing fused protein function, build plnk prokaryotic expression restructuring matter
Grain, named pET-32a-plnK.
3.pET-32a-plnK transfection, to BL21 (a) competent cell, optimizes abduction delivering condition, it is thus achieved that can be in vitro
The recombiant protein of abduction delivering.
4. purification of recombinant proteins, and carry out albumen checking.
5. Inhibition test confirms that this recombiant protein still has broad-spectrum antibacterial activity.
It is an advantage of the current invention that:
1. the present invention has been successfully established the gene engineering method that can carry out vivoexpression lactein plnK, and the method breaches
Wild strain cannot the shortcoming of great expression lactein.
2. the present invention still has the antibacterial of wide spectrum by the lactein plnK recombiant protein of gene engineering method gained
Effect, the method can be used for industrially scalable metaplasia lactic acid producing rhzomorph.
Accompanying drawing explanation
The PCR amplification of Fig. 1 lactein plnK gene, M:DNA molecular mass standard；1: lactein plnK base
Gene-amplification product.
The PCR of Fig. 2 positive recombinant expression plasmid identifies, M: DNA molecular mass standard；1:plnK upstream and downstream primer PCR
Product 2:T7 primer PCR product, 3:T7 downstream primer and purpose forward primer PCR primer；4: T7 forward primer and purpose are anti-
To primer PCR product.
The enzyme action of Fig. 3 recombinant expression plasmid is identified, M1, M2:DNA molecular mass standard；1: the pET-32a-of non-enzyme action
PlnK plasmid；2:pET-32a-plnK'sBamH ISingle endonuclease digestion；3:pET-32a-plnKSal ISingle endonuclease digestion；4:pET-32a-
plnK BamH IWithSal IDouble digestion.
Fig. 4 lactein plnK gene nucleotide series sequence analysis is analyzed.
Fig. 5 lactein plnK and the analysis of other bacterial strains plnK gene nucleotide series Phylogenetic tree.
Fig. 6 E.coliThe abduction delivering result of restructuring pET-32a-plnK albumen, M: protein molecule in BL21 (DE3)
Quality standard；1: blank group induction group；2:pET-32a (+) empty carrier bacterium induction group；3:pET-32a-plnK recombiant plasmid bacterium
Do not induce group；4:pET-32a-plnK recombiant plasmid bacterium induction group.
The optimization of Fig. 7 inducing temperature, M: protein standard；1: blank group induction group；2～7: be respectively 16
DEG C, 25 DEG C, 31 DEG C, 37 DEG C, the pET-32a-plnK recombiant plasmid bacterium group of induction under 43 DEG C of gradient temperatures.
The optimization of Fig. 8 induction time, M: protein standard；1: blank group induction group；2～9: be respectively induction
The pET-32a-plnK recombiant plasmid bacterium group of induction under rear 0 h, 4 h, 12 h, 16 h gradient.
The optimization of Fig. 9 IPTG induced concentration, M: protein standard；1: blank group induction group；2～9: respectively
It is 0 mmoL/L for IPTG induced concentration, 0.2 mmoL/L, 0.4 mmoL/L, 0.6 mmoL/L, 0.8 mmoL/L, 1.0
The pET-32a-plnK recombiant plasmid bacterium group of induction under mmoL/L, 1.2 mmoL/L, 1.5 mmoL/L gradient.
Figure 10 pET-32a-plnK recombinant protein purification result, M: protein standard；1: albumen is molten before purification
Liquid, 2～9: be respectively 40 mM, 80 mM, 120 mM, 160 mM, 200 mM, 240 mM, the imidazoles such as 280 mM and 320 mM is washed
The purification result of de-liquid
The BSA standard curve that Figure 11 BCA method is set up.
The Western-blot of Figure 12 pET-32a-plnK albumen analyzes, and His antibody is one to resist；1:pET-32a-plnK
Group；2:pET-32a (+) group;3: blank bacterium group.
Figure 13 recombiant protein changes antibacterial result, A: staphylococcus aureus B: escherichia coli C: Salmonella D: Pasteur's bar
Bacterium.
Detailed description of the invention
In order to make content of the present invention easily facilitate understanding, below in conjunction with detailed description of the invention to of the present invention
Technical scheme is described further, but the present invention is not limited only to this.
1 materials and methods
1.1 material
1.1.1(poison, bacterium) strain, laboratory animal and expression vector
Expressive host bacterium: E.coliDH5 α competent cell,E.coliBL 21 (DE3) competent cell is (purchased from Beijing
Quan Shijin biotech firm)；Expression vector: pET-32a (+) (purchased from Promega Beijing Bioisystech Co., Ltd).
1.1.2 main agents
High-purity plasmid is little carries middle amount test kit (DP107) (purchased from Tian Gen biochemical corp, Beijing)；Agarose gel DNA quickly returns
Receive test kit (CW2302) (being century biotech company purchased from Beijing health)；GoScripTMReverse Transcription
System and T4 ligase (purchased from Promega company)；BamH I QuickCut and andSal I QuickCut limits enzyme
(purchased from Dalian treasured biotech firm)；TransTaqTMDNA Polymerase High Fidelity(HiFi)(AP131-11)、
ProteinIsoTMNi-NTA Resin protein purification post (DP101-01), BCA determination of protein concentration test kit, Radix Cochleariae officinalis peroxidating
The goat-anti rabbit of thing enzyme (HRP) labelling and the anti-duck of rabbit two anti-and anti-His label protein one anti-(purchased from Beijing Quan Shi King Company)；
PageRuler Prestained Protein Ladder(10～180 ku), SuperSignal West Pico chemiluminescence
Substrate (purchased from Thermo Fischer Scient Inc.)；PAGE gel prepares test kit (AR0138), isopropyl-beta D-thio half
Lactoside (IPTG), Triton-X 100 and Phenylmethanesulfonyl fluoride (PMSF) are purchased from doctor moral Bioisystech Co., Ltd.
1.2 test method
1.2.1 the design of primer and synthesis
Lactic acid bacteria (accession number: X94434) the plnK full length gene sequence logged in reference to GenBank, uses PCrimer
A pair specific primer of Premier 6.0 software design, amplification total length 190 bp (174bp+16 bp restriction enzyme site), design
Primer sequence (as shown in table 1 below) be sent to Shanghai Li Fei biotech company by PAGE level purification process synthesize, TE buffer
Diluted concentration is that 10 μm oL/L are standby in-20 DEG C of freezings.
Table 1 plnK gene primer sequence table
1.2.2 the amplification of lactein plnK gene
1.2.2.1 the extraction of bacteria total DNA
Extracting DNA according to bacterial genomes DNA extraction kit description, concrete operation step is as follows:
(1) taking inoculum 1-5 ml, 10000r/min is centrifuged 1 min, and exhaust supernatant as far as possible；
(2) adding 200 μ L buffer GA in bacterial sediment, vibration to thalline thoroughly suspends；
(3) in pipe, 20 μ L Proteinase K solution are added, mixing.
(4) adding 220 μ L buffer GB, vibrate 15 sec, places 10 min for 70 DEG C, and solution strain is limpid, brief centrifugation
To remove the globule of cap wall.
(5) 220 μ L dehydrated alcohol are added, fully vibration mixing 15 sec, now it is possible that flocculent deposit, brief centrifugation
To remove the globule of cap wall.
(6) previous step gained solution and flocculent deposit are all added in an adsorption column CB3 that (adsorption column puts into collecting pipe
In), 12000 r/min are centrifuged 30 sec, outwell waste liquid, are put in collecting pipe by adsorption column CB3.
(7) the most first check whether before adding 500 μ L buffer GD(uses in adsorption column CB3 and added dehydrated alcohol),
12000 r/min are centrifuged 30 sec, outwell waste liquid, are put in collecting pipe by adsorption column CB3.
(8) the most first check whether before adding 600 μ L rinsing liquid PW(uses in adsorption column CB3 and added dehydrated alcohol),
12,000 r/min are centrifuged 30 sec, outwell waste liquid, and adsorption column CB3 puts in collecting pipe.
(9) repetitive operation step 8.
(10) putting back in collecting pipe by adsorption column CB3,12000 r/min are centrifuged 2 min, outwell waste liquid.By adsorption column
CB3 is placed in room temperature and places several minutes, thoroughly to dry rinsing liquid remaining in adsorbing material.
(11) adsorption column CB3 is proceeded in a clean centrifuge tube, the unsettled dropping 50-to the middle part of adsorbed film
200 μ L elution buffer TE, room temperature is placed 2-5 min, 12000 r/min and is centrifuged 2 min, collected by DNA solution centrifugal
Guan Zhong.
1.2.2.3 PCR amplification
GoScripTMReverse Transcription System transcriptive process,reversed and condition are with reference to GoTaq 2-Step
RT-qPCR System Kit explanation.Using upstream primers F and downstream primer R as amplimer, lactic acid bacteria STb gene is template,
Lactein plnK gene is carried out PCR amplification, and pcr amplification product agarose gel electrophoresis is identified, concrete PCR reaction system
With reaction condition (see table 2).
Table 2 PCR amplification system table and reaction condition
1.2.2 the structure of recombinant expression plasmid and qualification
1.2.2.1 the purification of pcr amplification product
(1) PCR primer is after agarose gel electrophoresis is identified correctly, cuts purpose band, utilizes glue to reclaim test kit and carries out pure
Change, glue is reclaimed the PCR primer of purification and is placed in-80 DEG C and saves backup.
1.2.2.2 the fragment of mesh is identified with the double digestion of expression vector
By ready pET-32a (+) prokaryotic expression carrier uses respectively with purpose fragment after purificationBamH IWithSal ICarry out
Double digestion reacts, reaction system and reaction condition (as shown in table 3 below and table 4), and after double digestion reaction terminates, glue reclaims pure again
Purpose fragment and the carrier segments of change enzyme action are in case being attached reaction.
The double digestion system of table 3 lactein plnK gene and condition
Table 4 pET-32a (+) the double digestion system of carrier and condition
1.2.2.3 coupled reaction
Take again fragment and the carrier segments of the PCR mesh of the enzyme action of glue recovery purification, carry out both agarose gel electrophoresis estimations
Concentration, is then attached reaction with purpose fragment and carrier molar ratio for 3:1, mole sees T with calculation method of physical volume4Connect
Enzyme description (promega company), linked system and reaction condition (referring to table 5 below):
Table 5 coupled reaction system and condition
1.2.2.4 recombinant plasmid transformed
Recombiant plasmid after connecting, converts and expands to DH5 α competent cell, and operating procedure is as follows:
(1) add connection product in 50 μ LDH5 α competent cells, after mixing, place 25 min on ice；
(2) after ice bath completes, 42 DEG C of heat shock 45 s, it is immediately placed on 2 min on ice；
(3) add 250 μ L and balance LB culture medium A mp to room temperature-, 200 r/min, 37 DEG C of oscillation incubations cultivate 1 h；
(4) take 200 μ L bacterium solution and be uniformly applied to the solid culture plate Amp containing LB+, it is inverted in 37 DEG C of constant incubators, mistake
(for obtain more clone, 4000 r/min are centrifuged 1 min, discard part supernatant, retain 100～150 μ L, gently in cultivation at night
Play even suspension thalline, take whole bacterium solution coated plate).
1.2.3.5 PCR identifies that recon and recombiant plasmid extract
Select white monoclonal in 10 μ L sterilized water, vortex mixed, carry out PCR with this mixed liquor for template and identify positive weight
Organize plasmid or expand with the T7 primer (primer sequence see table 6) on carrier.Same colony inoculation is contained in liquid simultaneously
Ammonia benzyl LB culture medium, 200 r/min, 37 DEG C of shaken cultivation are overnight.Utilize high-purity plasmid little carry middle amount test kit extract PCR
Positive plasmid DNA.
Table 6 T7 primer sequence table
1.2.2.6 the enzyme action of recombinant expression plasmid is identified and sequencing
Extracting PCR and identify that positive recombiant plasmid carries out double digestion qualification, enzyme action system is as shown in table 7 below, agarose gel electricity
PCR and endonuclease reaction product are identified in swimming, and qualification result meets intended recombiant plasmid and is sent to the survey of Shanghai Li Fei biotech company
Sequence by the named pET-32a-plnK of recombinant expression plasmid correct for sequencing result.
The enzyme action identification system of table 7 recombiant plasmid
1.2.4 the sequence analysis of lactein plnK gene
The nucleotide sequence comparison of NCBI website is utilized to analyze sequencing result；Use Mega biosoftware to carry out genetic homology simultaneously
The amino acid sequence analysis analyzed and derive.
1.2.5 recombiant plasmid pET-32a-plnK existsE.coliAbduction delivering in BL21 (DE3)
Recombiant plasmid pET-32a-plnK is converted extremelyE.coliIn BL21 (DE3) competent cell, the same 1.2.3.4 of method
Described, in flat board, picking white monoclonal colony inoculation is in the LB liquid medium of 1 mL, is placed in 200 r/min, 37 DEG C
Cultivation 4-6h in shaking table, takes a copy of it and adds the most final concentration of 1 mmoL/L induced protein expression of IPTG, and another part adds same
The distilled water of ratio as a control group, continues to be placed in shaking table and cultivates 24 h, set simultaneously pET-32a (+) empty carrier bacterium induction group
With blank induction group.The bacterium solution taking 300 μ L adds 5 × SDS-PAGE sample-loading buffer, boils after vibration mixing in boiling water
5min, cooled on ice 5min, in 4 DEG C, 12000 r/min are centrifuged 5min, take supernatant solution and carry out protein SDS-PAGE electrophoresis and divide
Analysis.Meanwhile, collect all of thalline, the resuspended washing of PBS 3 times, finally (add with the PBS of 1/10 volume
Protease inhibitor) resuspended thalline, it is placed under ultrasound wave and crushes thalline completely, collect bacterium solution and carry out SDS-PAGE electrophoretic analysis.
1.2.6 the SDS-PAGE electrophoretic analysis of pET-32a-plnK albumen
Draw 10 μ L above sample protein lysate respectively and carry out the SDS-PAGE electrophoretic analysis that gum concentration is 12%, SDS-PAGE
Gel configuration such as table 8 below, after electrophoresis terminates, takes out glue and is placed in 0.1% (W/V) coomassie brilliant blue R_250 staining solution dyeing 45
Min, carries out eluting in de-inking solution, take pictures after background decolouring is clean.
Table 8 PAGE gel preparation table
1.2.7 pET-32a-plnK protein induced expression system optimization
1.2.7.1 the optimization of the optimal inducing temperature of IPTG
The positive colony bacterium recovery that will preserve, is inoculated in LB culture medium, 200 r/min with the ratio of 1:100, trains in 37 DEG C of shaking tables
Support, add IPTG and make its final concentration of 1.0 mmol/L, be respectively placed in different thermograde 16 DEG C, 25 DEG C, 31 DEG C, 37 DEG C,
Carry out abduction delivering under the conditions of 43 DEG C, take 10 μ L protein samples and carry out SDS-PAGE electrophoresis.
1.2.7.2 the optimization of the optimal induction time of IPTG
Under the conditions of optimal inducing temperature, it is ensured that under the final concentration of 1.0 mmoL/L condition of culture of IPTG are constant, respectively in difference
Point in time sampling: 0h, 4 h, 8 h, 12 h, 16 h, take the 10 above protein samples of μ L and carry out SDS-PAGE electrophoresis.
1.2.7.3 the optimization of the optimal induced concentration of IPTG
On the basis of optimizing the optimal inducing temperature that obtains and incubation time, it is separately added into the IPTG derivant of different final concentration: 0
MmoL/L, 0.2 mmoL/L, 0.4 mmoL/L, 0.6 mmoL/L, 0.8 mmoL/L, 1.0 mmoL/L, 1.2 mmoL/L, 1.5
MmoL/L induces, and finally determines optimal induced concentration.Sampling and SDS-PAGE step see 1.2.5 and 1.2.6.
1.2.8 the purification of pET-32a-plnK albumen and determination of protein concentration
1.2.8.1 the Ni-NTA affinitive layer purification of pET-32a-plnK albumen
200 r/min, mass propgation positive bacteria by after conditions above abduction delivering in 37 DEG C of shaking tables, collect thalline, PBS buffers
Liquid washs 3 times, and brotein equilibrium buffer hangs again with 1/10 volume, and ultrasound wave crushes thalline, centrifuging and taking supernatant conduct completely
Sample to be purified.According to ProteinIsoTM Ni-NTA Resin operational approach carries out separation and purification of protein, by variable concentrations miaow
Azoles (40 mmoL/L, 80 mmoL/L, 120 mmoL/L, 160mmoL/L, 200 mmoL/L, 240 mmoL/L, 280 mmoL/L,
320 mmoL/L) the collection liquid of eluting carries out SDS-PAGE electrophoretic analysis, determines the optimal eluting concentration of imidazoles.
ProteinIsoTMNi-NTA Resin carries out isolated and purified process to His label protein and generally includes: dress post, balance, on
The steps such as sample, washing, eluting, regeneration are as follows:
(1) dress post: resuspended absorption filler (protein load 10～20 mg/mL), stands in adding chromatographic column to post height about chromatography
2/3 volume of post；
(2) balance: the balance liquid buffer balance chromatographic column of 5～10 times of chromatographic column volumes, for the restructuring egg that binding ability is strong
In vain, low concentration imidazoles (10～20 mmoL/L) can be added to improve specific binding raising purification efficiency；
(3) loading: protein sample buffer keeps consistent with level pad as far as possible.Protein sample solution can by centrifugation or micro-
Filter (0.45 μm oL/L) processes in order to avoid blocking chromatographic column；
(4) washing: the equilibration buffer solution chromatographic column of 5～10 times of column volumes, collects effluent；
(5) albumen eluting: the imidazoles eluting destination protein of preparation level pad preparation variable concentrations, collects effluent；
(6) chromatographic column reclaims: first with the equilibration buffer solution of 5～10 times of volumes, then add 5～10 times of volume milli-Q water,
Finally by 5 times of volume 75% washing with alcohol, finally take out filler and be stored in chromatographic column buffer.Milli-Q water pillar is dried guarantor
Deposit.
1.2.8.2 the concentration of albumen measures after purification
Use BCA protein concentration quantitative test box, detect and draw standard curve by microplate reader, calculate the pET-32a-of purification
PlnK protein concentration, Detailed operating procedures is as follows:
(1) BSA titer: 1 × PBS is by BSA Standard SolutionA(25 mg/mL) to be diluted to concentration be 0.5 mg/
mL；
(2) BCA working solution: according to sample number, BCA Solution A and BCA Solution B is pressed the dilution proportion of 50:1
Becoming BCA working solution, vibration mixing is standby；
(3) in the sample well in 96 clean holes, adding 0 with this, after 1,2,4,8,12,16,20 μ L dilution, concentration is 0.5
The BSA titer of mg/mL, 1 × PBS complements to the final volume of every hole 20 μ L；
(4) testing sample is added in 96 holes by the preparation of testing sample: 1 × PBS according to after certain dilution proportion；
(5) in each sample well, add the BCA working solution of 200 μ L, put 37 DEG C, 30-90 min, 60 DEG C, 30 min or room
Temperature 2 h；
(6) 96 orifice plates are placed under 562 nm wavelength with microplate reader detection, draw standard curve, and calculate testing protein sample
Concentration value.
1.2.9 the Western blot of pET-32a-plnK albumen identifies
By recombiant protein pET-32a-plnK after purification after SDS-PAGE is separated by electrophoresis, immunoblotting checking (Western
Blot) albumen is the most correct.Concrete operation step is as follows:
1.2.9.1 transferring film
(1) take off PAGE glue, prevent glue to be dried, according to target protein molecular weight, cut required gel with reference to albumen Marker, use
Appropriate transfering buffering liquid balanced gel 30 min；
(2) transfer is folded up in bigger pallet, add enough transfering buffering liquids to not having whole transfer blade；
(3) according to cutting glue size 6 filter paper of shearing and 1 NC film or (pvdf membrane)；The transfer liquid moistening in advance of NC film, PVDF needs
First soak with methanol；
(4) transfer interlayer order is by the filter paper of 3 layers of moistening, and PAGE glue, film, three metafiltration paper are carried out, and often fold one layer, need to first use glass
Rod is rushed bubble.
(5) close transfer folder, it is ensured that both positive and negative polarity, transfer is folded up into transfer groove, adds film transfering buffering liquid, adjusts neat transfer
Buffer level, connects power supply, and (10-20 DEG C) 100 V electricity turns 2 h in the cooling condition；
(6) take out film, wash 2 times with TBST, remove transfering buffering liquid.
1.2.9.2 antibody incubation
(1) close: the defatted milk powder confining liquid of 10% is closed overnight in 4 DEG C of refrigerators；
(2) washing: TBST buffer solution 3 times, each about 5 min；
(3) one anti-hatch: the defatted milk powder confining liquid dilution goat-anti HIS positive serum of 10%, in 4 DEG C of refrigerator overnight incubation；
(4) washing: TBST buffer solution 3 times, each about 5 min；
(5) two anti-hatch: the rabbit anti-sheep IgG antibody of the defatted milk powder confining liquid dilution HRP labelling of 10%, hatch 2 h for 37 DEG C；
(6) washing: TBST buffer solution 3 times, each about 5 min.
(7) ECL chemiluminescence detection colour developing, exposure, take pictures.
The external bacteriostatic experiment of recombiant protein
Selecting four kinds of indicator bacterias is staphylococcus aureus respectively, escherichia coli, and Salmonella, pasteurellosis bacillus, Odontothrips loti is tested
Demonstrate,prove the bacteriostatic activity of the recombiant protein of external prokaryotic expression gained.Staphylococcus aureus, escherichia coli, Salmonella is used respectively
Ordinary nutrient agar culture medium, the pasteurellosis bacillus Martin's culture medium culturing adding 10% Ox blood serum.Recombiant protein usage amount 200 μ L
(0.5mg/mL)
2 results and analysis
The PCR amplification of 2.1 lactein plnK genes
Through nucleic acid electrophoresis, pcr amplification product identifies that a visible size is about the specific band (as shown in Figure 1) of 174bp, with
Intended clip size is consistent, it is seen that designed primer correctly can use.
The qualification result of 2.2 recombinant expression plasmids
Coated plate after Transfected Recombinant Plasmid DH5 α, selecting white monoclonal bacterium colony is template, enters with upstream primers F and downstream primer R
Performing PCR is identified, positive bacterium amplifiable go out a specific band being about 190bp；With carrier universal primer T7 amplifiable
Bar height is about the purpose band of 700 bp sizes, result as in figure 2 it is shown, and then extract its plasmid and carry out enzyme action qualification, includingBamH IWithSal I double digestion,BamH ISingle endonuclease digestion, Sal I single endonuclease digestion result is illustrated in fig. 3 shown below, and the product of single endonuclease digestion is visible
Article one, size is about the linear plasmid band of about 6090 bp, has more a band being about 190 bp seen from double digestion, with
Intended size is consistent, and tentatively shows construction of recombinant expression plasmid success.Sequencing result proves that recombiant plasmid is by success further
Build.
2.3 the sequencing results
The lactein plnK gene order delivered in result order-checking obtained and Genebank carries out nucleotide sequence comparison,
Find that the 8 strains of lactic acid bacteria strains that this lactic bacteria strain and Genebank issue have the homology of more than 96%, wherein with Yunnan bacterial strain 5-
2, Beijing bacterial strain CAUH2, Algeria bacterial strain LbM2a, Zhejiang bacterial strain LZ95, Hangzhou bacterial strain LZ206, Sichuan bacterial strain ZS2058
Homology is 100%；Wherein with China bacterial strain L-XM1, L-ZS9 homology be 96%.Through these bacterial strains are carried out cladogram
Analyze, find that this lactic bacteria strain and Yunnan bacterial strain 5-2 belong to same hypotype；With Beijing bacterial strain CAUH2, Algeria's bacterial strain
LbM2a, Hangzhou bacterial strain LZ206, Zhejiang bacterial strain LZ95, Sichuan bacterial strain ZS2058 belong to same type；With China bacterial strain L-XM1, L-
ZS9 belongs to different shaped.
The abduction delivering result of 2.4 pET-32a-plnK recombiant proteins
The expection albumen size that recombinant expression carrier gives expression to is about 26.2ku.SDS-PAGE result shows (as shown in Figure 6),
PET-32a-plnK recombiant plasmid bacterium is after IPTG induces, and high-visible size is about the specific band of 26.2ku, blank bacterium
Induction group and pET-32a (+) empty carrier bacterium induction group is showed no this specific band, and the pET-32a-plnK of induction recombinates matter
Grain induction group band brightness is significantly greater than the pET-32a-plnK recombiant plasmid group do not induced, additionally, pET-32a (+) empty carrier
Bacterium induction group is compared with blank bacterium, it is seen that a size is about the band of 20 ku, and these are also many with intended empty carrier label protein
Peptide size is consistent.
The optimum results of 2.5 pET-32a-plnK recombiant protein abduction delivering conditions
2.5.1 the optimization of the optimal inducing temperature of IPTG
Under conditions of final concentration of 1.0 mmoL/L of IPTG, pET-32a-plnK recombiant plasmid bacterium is induced 24 h, point
Analyse 16 DEG C, 25 DEG C, 31 DEG C, 37 DEG C, the pET-32a-plnK expression of recombinant proteins amount of induction, result at a temperature of 43 DEG C
Being illustrated in fig. 7 shown below, destination protein expression in the thermograde of 16～37 DEG C becomes larger, and expresses under conditions of 37 DEG C
Amount reaches maximum, and after 43 DEG C, expressing quantity gradually decreases,
2.5.2 the optimization of the optimal induction time of IPTG
At 37 DEG C, under conditions of final concentration of 1.0 mmoL/L of IPTG, analyze pET-32a-plnK recombiant plasmid bacterium and exist respectively
Expressing quantity change under the different induction times such as 0 h, 4 h, 8 h, 12 h, 16 h.Result shows (as shown in Figure 8),
PET-32a-plnK recombiant plasmid bacterium, when inducing 4h, has begun to express destination protein, and the expression of destination protein is at any time
Between passage gradually step up, until induction 12 h after reach maximum, hereafter, the expression of albumen gradually tends to balance.
2.5.3 the optimization of the optimal induced concentration of IPTG
At 37 DEG C, induction time is under the inductive condition of 12 h, respectively at IPTG final concentration of 0 mmoL/L, 0.2 mmoL/L,
0.4 mmoL/L, 0.6 mmoL/L, 0.8 mmoL/L, 1.0 mmoL/L, 1.2 mmoL/L, lure under the gradient of 1.5 mmoL/L
Leading, result is as it is shown in figure 9, the expression of the final concentration of 0 mmoL/L albumen of IPTG is little, and concentration is 0.2～1.0 mmoL/L's
Time between gradient, the expression of albumen has the trend being gradually incremented by, and reaches maximum when the concentration of 0.4 mmoL/L,
The Ni-NTA affinitive layer purification result of 2.6 pET-32a-plnK recombiant proteins
Bacterium solution after ultrasonication passes through Ni-NTA affinity column, carries out washing pure respectively with the imidazoles diluent of variable concentrations
Changing, and carry out SDS-PAGE analysis, result shows (such as Figure 10), and the protein band obtained after eluting is more and more single, says
Bright nonspecific protein impurities significantly reduces, and the eluent efficiency with the imidazole concentration of 200 mM is the highest.Pass through BCA
It is 0.5 mg/mL that method (as shown in figure 11) calculates protein concentration after purification.
The Western-blot qualification result of 2.7pET-32a-plnK recombiant protein
Western blot analysis result shows (as shown in figure 12), pET-32a-plnK recombiant plasmid bacterium group and pET-32a (+)
Empty carrier bacterium group is about 26.2 ku and 20 ku in size respectively and a specific band occurs, and this does not occurs in blank bacterium group
Band.Result shows that pET-32a-plnK recombiant protein is desired albumen, and destination protein is active.
The 2.7 antibacterial results of pET-32a-plnK recombiant protein
Bacteriostatic experiment result shows, recombiant protein pET-32a-plnK remains able to effectively suppress staphylococcus aureus, greatly
Enterobacteria, Salmonella, pasteurellosis bacillus (see Figure 13).
The foregoing is only presently preferred embodiments of the present invention, all impartial changes done according to scope of the present invention patent with
Modify, all should belong to the covering scope of the present invention.
SEQUENCE LISTING
<110>University Of Agriculture and Forestry In Fujian
<120>a kind of gene engineering method improving lactein plnK yield
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 28
<212> DNA
<213>artificial sequence
<400> 1
gcggatccat gaaaattaaa ttaactgt 28
<210> 2
<211> 28
<212> DNA
<213>artificial sequence
<400> 2
gcgtcgactc acttattata atcccttg 28
<210> 3
<211> 20
<212> DNA
<213>artificial sequence
<400> 3
taatacgact cactataggg 20
<210> 4
<211> 19
<212> DNA
<213>artificial sequence
<400> 4
gctagttatt gctcagcgg 19
Claims (3)
1. the primer being used for improving lactein plnk throughput method, it is characterised in that: described primer sequence is: upstream
Primers F: 5 '-GCGGATCCATGAAAATTAAATTAACTGT-3 '；Downstream trip primer R:5 '-
GCGTCGACTCACTTATTATAATCCCTTG-3’。
2. the gene engineering method improving lactein plnK yield, it is characterised in that: recombinate by building genes of interest
Expression plasmid, optimization expression system, it is thus achieved that can the technique for gene engineering of great expression lactein plnK in vitro.
A kind of gene engineering method improving lactein plnk yield the most according to claim 2, it is characterised in that: institute
The method of stating comprises the steps:
1) designing a pair plnk specific primer, primer sequence is forward primer F:5 '-GCGGATCCATGAAAATTAAATTAAC
TGT-3’；Downstream trip primer R:5 '-GCGTCGACTCACTTATTATAATCCCTTG-3 '；
2) select that there is the excellent carrier pET-32a(+ expressing fused protein function), build plnK prokaryotic expression restructuring matter
Grain, named pET-32a-plnK；
PET-32a-plnK transfection to competent cell, optimizes abduction delivering condition, it is thus achieved that can the weight of abduction delivering in vitro
Histone；
Purification of recombinant proteins, and carry out albumen checking；
Inhibition test confirms that this recombiant protein still has broad-spectrum antibacterial activity.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109134628A (en) * | 2018-09-26 | 2019-01-04 | 福建傲农生物科技集团股份有限公司 | A kind of lactein plnE and preparation method thereof |
| CN109170268A (en) * | 2018-09-26 | 2019-01-11 | 江苏傲农生物科技有限公司 | A kind of application of lactein plnE |
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