CN101948856A - Recombinant lactobacillus rhamnosus engineering strain and preparation method thereof - Google Patents
Recombinant lactobacillus rhamnosus engineering strain and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a recombinant lactobacillus rhamnosus engineering strain and a preparation method thereof. The lactobacillus rhamnosus engineering strain carries expression vectors of a catalase gene and a superoxide dismutase gene which are connected in series. The method for preparing the engineering strain comprises the following steps of: 1) amplifying catalase gene segments and superoxide dismutase gene segments from genomes of lactobacillus sake and streptococcus thermophiles by a polymerase chain reaction (PCR) method; 2) designing a specific restriction enzyme site, wherein the restriction enzyme site is connected in series with an escherichia coli-lactobacillus shuttle plasmid vector pSIP502; and 3) transforming a recombinant plasmid into lactobacillus rhamnosus by an electroporation method. The recombinant lactobacillus rhamnosus engineering strain prepared by the method can remarkably enhance oxidation resistance and can be applied to the fermented food industry.
Description
Technical field
The present invention relates to the microorganism biology field, be specifically related to reorganization lactobacillus rhamnosus engineering strain that improves resistance of oxidation and preparation method thereof.
Background technology
(Lactic acid bacteria is the general term that can fermenting carbohydrate produces the gram positive bacterium of lactic acid LAB) to milk-acid bacteria, is internationally recognized one big class probiotic bacterium, and lactobacillus-fermented product has become topmost functional foodstuff.The milk-acid bacteria great majority belong to facultative anaerobic bacteria, and process of growth does not generally need oxygen, and the existence of oxygen may work the mischief to it.Oxidative stress mainly comes from various active oxygen kinds, and (Reactive oxygen spicies ROS), comprises superoxide anion (O
2 -), hydrogen peroxide (H
2O
2), hydroxyl radical free radical (OH) etc., ROS can destroy biomacromolecules such as protein, nucleic acid, cytolemma, causes the cell decline, wherein OH hazardness maximum.
During evolution, various antioxidant reductases have appearred in the bacterial cell, wherein the most important thing is superoxide-dismutase (Superoxide dismutase, SOD) and catalase (Catalase, CAT), the former the O that can degrade
2 -, the latter can remove H
2O
2, finally stop the accumulation of OH, the not harm of oxidated pressure of protection cell.Yet most of lactobacillus lack superoxide dismutase gene, have only the minority bacterial classification to contain catalase gene, thereby a little less than the resistance to oxidation pressure.
Studies show that, lactobacillus rhamnosus is a kind of Bacterium lacticum that benefit is given birth to function that typically has, do not contain CAT and sod gene on its karyomit(e), the anti-oxidant ability of coercing a little less than, as a kind of functional lactobacillus commonly used, in the leavened prod course of processing, can be subjected to the harm of various oxidizing conditions, cause deterioration in quality.
In recent years, improve the technology develop rapidly of F-strain resistance of oxidation by the method that imports foreign gene.CAT gene and sod gene that people will come from plant lactobacillus, lactobacillus sake, Bacillus subtilus and the thermophilus streptococcus change in the weak milk-acid bacteria acceptor of various oxygen-resistant abilities, successfully improved the antioxidant levels of these milk-acid bacterias, in lactobacillus rhamnosus, but do not reported.
Summary of the invention
The purpose of this invention is to provide a kind of recombinant vectors, carry bacterium catalase gene and superoxide dismutase gene, can give expression to catalase and superoxide-dismutase.
Described catalase gene clone is from lactobacillus sake, and described superoxide dismutase gene clone is from thermophilus streptococcus.
Described superoxide dismutase gene also comprises himself promotor.
The primer of described pcr amplification catalase gene is 5 '-CATGCCATGGCAAATCAACTAACGACT-3 ' and 5 '-CCGCTCGAGAAAATAGGTGTCCCAAAC-3 '; The primer of described pcr amplification superoxide dismutase gene is 5 '-CCGCTCGAGCAAGATTTTGTAAG-3 ' and 5 '-GGGGTACCTGAGGATGATTCTAGAC-3 '.
Another object of the present invention provides a kind of reorganization lactobacillus rhamnosus engineering strain, and this bacterial strain contains the recombinant vectors that can express peroxidation hydrogenation enzyme and superoxide-dismutase.
Another object of the present invention provides a kind of preparation method of the lactobacillus rhamnosus engineering strain of recombinating, and comprises the steps:
1) amplification catalase gene and the superoxide dismutase gene fragment from the genome of lactobacillus sake and thermophilus streptococcus respectively of the method by PCR;
2) series connection of catalase gene that amplification is obtained and superoxide dismutase gene is building up on intestinal bacteria-Bacterium lacticum shuttle vector pSIP502 (Sorvig et al., 2003), obtains recombinant plasmid;
3), cultivate intestinal bacteria and extract plasmid with the recombinant plasmid transformed intestinal bacteria;
4) by electroporation recombinant plasmid transformed is arrived lactobacillus rhamnosus AS 1.2466.Wherein lactobacillus rhamnosus AS 1.2466 is the reference culture of Chinese common micro-organisms culture presevation administrative center Institute of Microorganism, Academia Sinica preservation, and preserving number is CGMCC 1.2466T.
Another object of the present invention provides the application of described reorganization lactobacillus rhamnosus on fermented food industry.
The lactobacillus rhamnosus that katA of containing provided by the invention and sodA integrate recombinant plasmid altogether can significantly improve the anti-H of this lactobacillus rhamnosus
2O
2Toxic ability.Through measuring, change the katA recombinant plasmid over to and change katA over to and sodA integrates the lactobacillus rhamnosus of recombinant plasmid altogether at 6mM H
2O
2The survival rate of handling behind the 1h is respectively 81.5% and 100%, exceeds nearly 1,000 times than the survival rate of the lactobacillus rhamnosus that contains the blank plasmid; And change the H of the lactobacillus rhamnosus of cointegrating plasmid pSIPCS at greater concn
2O
2Survival rate after handling exceeds more than 100 times again than the lactobacillus rhamnosus that only contains CAT.Simultaneously, change the lactobacillus rhamnosus that katA and sodA integrate recombinant plasmid altogether over to, can under the culture condition of ventilation more viable count be arranged, the fate of survival is also longer, has improved resistance of oxidation greatly.
Description of drawings
Figure 1 shows that the structure synoptic diagram of recombinant plasmid pSIPCAT and pSIPCS.A among the figure: intestinal bacteria-Bacterium lacticum shuttle plasmid pSIP502; B: recombinant plasmid pSIPCAT; C: integrate recombinant plasmid pSIPCS altogether.
Figure 2 shows that recombinant plasmid pSIPCS double digestion is identified in the lactobacillus rhamnosus.A identifies the katA gene for the NcoI/XhoI double digestion among the figure.M, DL15000; 1, pcr amplification katA gene; 2, the double digestion of plasmid pSIPCS.B identifies the sodA gene for the XhoI/KpnI double digestion.M, DL15000; 1, pcr amplification sodA gene; 2, the double digestion of plasmid pSIPCS.
Figure 3 shows that the detection of reorganization lactobacillus rhamnosus thalline degraded hydrogen peroxide.1 for changing the lactobacillus rhamnosus thalline of control vector pSIPCK among the figure; 2 for changing the lactobacillus rhamnosus thalline of recombinant plasmid pSIPCAT over to; 3 for changing the lactobacillus rhamnosus thalline of common integration recombinant plasmid pSIPCS over to.
Figure 4 shows that reorganization lactobacillus rhamnosus growing state under the long-term aeration condition.represents to contain the lactobacillus rhamnosus of recombinant plasmid pSIPCAT among the figure; ◆ expression contains the lactobacillus rhamnosus of control vector pSIPCK.
Figure 5 shows that reorganization lactobacillus rhamnosus growing state in the low sugar culture-medium.△ represents to contain the lactobacillus rhamnosus of common integration recombinant plasmid pSIPCS among the figure; ◇ represents to contain the lactobacillus rhamnosus of recombinant plasmid pSIPCAT.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment.
1.1 lactobacillus sake extracting genome DNA
Genome extracts and adopts bacterial genomes DNA extraction test kit (TIANGEN Biotech (Beijing) Co., Ltd.), and method is as follows:
(1) get the lactobacillus sake bacterium liquid 5ml that is cultured to logarithmic phase, centrifugal 1 minute of 12000rpm, supernatant discarded, it is once resuspended to add 200 μ l TES, and centrifugal 1 minute of 12000rpm inhales as far as possible and removes supernatant;
(2) add 180 μ l N,O-Diacetylmuramidases (20mg/ml), 37 ℃ of water bath processing 1 hour;
(3) add 20 μ l RNase (10mg/ml), slightly shook 15 seconds, room temperature was placed 5 minutes;
(4) add 20 μ l Proteinase K solution, mixings gently;
(5) add 220 μ l damping fluid GB, vibrate 15 seconds, 70 ℃ of water bath processing 10 minutes, brief centrifugal removal pipe covers and the globule of inwall;
(6) add 220 μ l dehydrated alcohols, fully mixing is 15 seconds, and is briefly centrifugal;
(7) previous step gained solution and flocks are transferred among the adsorption column CB3, put collection tube, centrifugal 30 seconds of 12000rpm outwells waste liquid;
(8) add 500 μ l damping fluid GD in adsorption column, centrifugal 30 seconds of 12000rpm outwells waste liquid;
(9) add 700 μ l rinsing liquid PW in adsorption column, centrifugal 30 seconds of 12000rpm outwells waste liquid, adds 500 μ l rinsing liquid PW washing more once;
(10) adsorption column is put back in the collection tube, centrifugal 2 minutes of 12000rpm outwells waste liquid, places room temperature to place several minutes, thoroughly dries the remaining rinsing liquid in the sorbing material;
(11) adsorption column is changed in the clean centrifuge tube, to the elution buffer TE of the unsettled Dropwise 50 μ l in the middle part of adsorption film through 65-70 ℃ of preheating, room temperature was placed 5 minutes, centrifugal 2 minutes of 12000rpm, solution is collected centrifuge tube, agarose gel electrophoresis Detection and Extraction result, and be stored in-20 ℃ standby.
1.2PCR amplification catalase gene fragment katA
Upstream primer: 5 '-CATG
CCATGGCAAATCAACTAACGACT-3 ';
Downstream primer: 5 '-CCG
CTCGAGAAAATAGGTGTCCCAAAC-3 '.
In the upstream and downstream primer respectively primer Nco I and Xho I restriction enzyme site, i.e. the part of band horizontal line in the sequence.
The PCR response procedures:
Component dosage (unit: μ l)
Lactobacillus sake genomic dna 1
Upstream primer (10 μ M) 0.5
Downstream primer (10 μ M) 0.5
dNTPs(10mM?each) 0.5
Ex Taq enzyme 0.5
10 * reaction buffer 2
Aseptic double-distilled water 15
The PCR reaction conditions:
95 ℃ of pre-sex change 5min, 94 ℃ of sex change 60s, 58 ℃ of annealing 30s, 72 ℃ are extended 60s, and 72 ℃ are extended 10min, totally 30 circulations eventually.
1.3katA be connected to plasmid pSIP502
PCR product and plasmid pSIP502 are through Nco I and Xho I double digestion, and the enzyme system of cutting sees the following form:
37 ℃ of enzymes are cut 4h.Through agarose gel electrophoresis, reclaim the purpose fragment of katA PCR product and the carrier segments of pSIP502.
The pSIP502 carrier segments is connected with katA fragment according to the form below system:
Component dosage (μ l)
Enzyme is cut back pSIP502 1
Enzyme is cut back katA fragment 5
10 * reaction buffer 1
Aseptic double-distilled water 2
4 ℃ of connections are spent the night, and will connect product transformed into escherichia coli DH5 α.Carry out as follows: get the bacillus coli DH 5 alpha competent cell, ice bath 30min melts, and draws 40 μ l competent cells and is connected product mixing, 42 ℃ of thermal shock 90s with 5 μ l, ice bath 2min immediately, add the fresh LB substratum of 1ml, 1.5h is cultivated in 37 ℃ of recoveries, gets an amount of bacterium liquid afterwards and coats the LB flat board that contains erythromycin (300 μ g/ml), picking list bacterium colony behind 37 ℃ of constant incubators cultivation 24h, PCR identifies recon, recon called after pSIPCAT, and the plasmid of extraction recon.
Simultaneously, the pSIP502 behind the double digestion carry out flush endization, connects, and makes up control plasmid pSIPCK, carries out as follows:
(1) the terminal flush endization of pSIP502 carrier viscosity behind the double digestion
Behind NcoI and XhoI double digestion, use dna polymerase i (the big fragment of Klenow) with the sticky end polishing, form blunt end.Reaction system comprises: the pSIP502 carrier segments 5 μ l after enzyme is cut, dNTPs 0.5 μ l, the big fragment 1 μ l of Klenow, 10 * reaction buffer, 2 μ l, aseptic double-distilled water 11.5 μ l, cumulative volume 20 μ l.Reaction conditions is: 37 ℃, and 2 hours.
(2) carrier flush end connection
Reclaim the carrier segments of flush endization, use T
4Dna ligase is connected to form empty carrier pSIPCK.
Reaction system comprises: the carrier segments 8 μ l of flush endization, T
4Dna ligase 1 μ l, 10 * reaction buffer, 1 μ l, cumulative volume 10 μ l.Reaction conditions is: 4 ℃, connection is spent the night.
Connect product and carry out the conversion of bacillus coli DH 5 alpha by the method for embodiment 1, the empty carrier called after pSIPCK that obtains.
2.1 extract the genomic dna of thermophilus streptococcus
Method is with embodiment 1.
2.2PCR amplification catalase gene fragment sodA
Upstream primer: 5 '-CCG
CTCGAGCAAGATTTTGTAAG-3 '
Downstream primer: 5 '-GG
GGTACCTGAGGATGATTCTAGAC-3 '.
Introduce Xho I and Kpn I restriction enzyme site in the upstream and downstream primer respectively, promptly drawn the horizontal line part in the sequence.
The PCR program is as follows:
Component dosage (unit: μ l)
The genomic dna 1 of thermophilus streptococcus
Upstream primer (10 μ M) 0.5
Downstream primer (10 μ M) 0.5
dNTPs(10mM) 0.5
Ex Taq enzyme 0.5
10 * reaction buffer 2
Aseptic double-distilled water 15
The PCR reaction conditions is as follows:
95 ℃ of pre-sex change 5min, 94 ℃ of sex change 60s, 56 ℃ of annealing 30s, 72 ℃ are extended 40s, and 72 ℃ are extended 10min, totally 30 circulations eventually.
2.3sodA be connected to plasmid pSIPCAT
The PCR product of sodA and plasmid pSIPCAT are through Xho I and Kpn I double digestion, and the enzyme system of cutting sees the following form:
37 ℃ of enzymes are cut 4h.Through agarose gel electrophoresis, the purpose fragment of the PCR product of recovery sodA and the carrier segments of pSIPCAT.
The pSIPCAT carrier segments is connected with sodA fragment according to the form below system:
Component dosage (μ l)
Enzyme is cut back pSIPCAT 1
Enzyme is cut back sodA fragment 7
10 * connection damping fluid 1
Aseptic double-distilled water 0
4 ℃ of connections are spent the night.Connect product and carry out the conversion of bacillus coli DH 5 alpha by the method for embodiment 1, recombinant plasmid called after pSIPCS.
3.1 preparation lactobacillus rhamnosus competence
With MRSS (MRS, 0.3M sucrose, 1% glycine (W/W)) culture medium culturing lactobacillus rhamnosus, grow to OD
600=0.4～0.6, get 10ml bacterium liquid, in 6000rpm, 4 ℃ of centrifugal 8min collect thalline; Add 2ml rinsing liquid (0.3M sucrose, 1mM MgCl
2) resuspended 2 times, 6000rpm, 4 ℃ of centrifugal 8min, supernatant discarded; Add the resuspended thalline of 2ml 30% (W/W) PEG-1500,6000rpm, 4 ℃ of centrifugal 10min, supernatant discarded is used with the resuspended back of 200 μ l30%PEG-1500.
3.2 the recombinant plasmid electricity transforms lactobacillus rhamnosus
Get lactobacillus rhamnosus competent cell 40 μ l, mix, be transferred in the 2mm electricity revolving cup, use Bio-Rad Gene Pulser Xcell with 2 μ l recombinant plasmid pSIPCS
TMType electricity conversion instrument, electric conversion condition is: 1.5kv/cm, 9ms.The electric shock back adds 1ml recovery substratum (MRS, 0.3M sucrose, 0.1M MgCl
2) in 37 ℃ of cultivation 2h, get an amount of bacterium liquid and coat the MRS flat board that contains erythromycin (5 μ g/ml), behind 37 ℃ of cultivation 48h, screen recon.
3.3 the evaluation of reorganization lactobacillus rhamnosus
Contain picking list bacterium colony on the MRS flat board of erythromycin, transfer in the liquid MRS substratum that contains erythromycin (5 μ g/ml), be cultured to stationary phase, extract plasmid, choose NcoI, XhoI and carry out double digestion evaluation katA gene, XhoI, KpnI double digestion are identified sodA gene (Fig. 2).
CAT is active in the embodiment 4 reorganization lactobacillus rhamnosus detects
4.1 utilize bubble method to detect the CAT enzymic activity
Switching reorganization lactobacillus rhamnosus is cultured to logarithmic phase (OD to the MRS substratum that contains protoheme (30 μ mol/L)
600Be 0.8) results, get 1ml bacterium liquid, the centrifugal 5min of 12000r/min, supernatant discarded with the resuspended thalline of 30 μ l TES (25% sucrose (W/W), pH 8.0 for 50mM Tris-Cl, 1mM EDTA), is got the resuspended liquid of 20 μ l and is mixed with 10 μ l30% hydrogen peroxide (W/W); The catalatic existence H that can degrade
2O
2Generate H
2O and O
2, produce bubble.
The result shows, changes lactobacillus rhamnosus thalline and the high-strength hydrogen peroxide mixed solution of control vector pSIPCK over to, no bubble; Change lactobacillus rhamnosus thalline and the high-strength hydrogen peroxide mixed solution of recombinant plasmid pSIPCAT over to, produce a large amount of bubbles; Change lactobacillus rhamnosus thalline and the high-strength hydrogen peroxide mixed solution of common integration recombinant plasmid pSIPCS over to, produce a large amount of bubbles (Fig. 3).Above-mentioned test all repeats 5 clones, each clone's effect unanimity.
4.2 recombinant C AT enzyme assay
Connect reorganization lactobacillus rhamnosus to the MRS cultivation that contains protoheme and concentrate, be cultured to the logarithmic phase results, get 1ml bacterium liquid, the centrifugal 5min of 12000r/min, supernatant discarded, resuspended with 6ml phosphoric acid buffer (0.1M, pH 8.0), resuspended liquid and 4ml 0.2M H
2O
2Mix, reaction 10min, per minute is got the 1ml mixed solution, with 2ml K
2Cr
2O
7/ HAc (5%K
2Cr
2O
7(W/W): HAc=1: 2) mix, boiling water bath reaction 10min is cooled to the centrifugal 5min of 12000r/min after the room temperature, gets supernatant and measures light absorption value in 570nm.
H with the 1ml different concns
2O
2With 2ml K
2Cr
2O
7570nm place light absorption value is measured in/HAc reaction, draws light absorption value-H
2O
2Concentration standard curve.
After measuring light absorption value, utilize typical curve to calculate degraded H in the resuspended liquid unit time of thalline
2O
2Amount, be enzyme activity, unit: μ mol H
2O
2/ min/10
8C.f.u..
Is 0 through detecting lactobacillus rhamnosus AS 1.2466 wild-types all with the CAT activity that contains the lactobacillus rhamnosus of control plasmid pSIPCK, and the CAT activity that changes the lactobacillus rhamnosus AS 1.2466 of recombinant plasmid pSIPCAT over to is 2.11 μ mol H
2O
2/ min/10
8C.f.u., the CAT activity that changes the lactobacillus rhamnosus of common integration recombinant plasmid pSIPCS over to is 2.53 μ molH
2O
2/ min/10
8C.f.u..Above-mentioned data all are the mean value that the test through 5 clones records.
The active detection of SOD of embodiment 5 reorganization lactobacillus rhamnosus
5.1 the mensuration of total protein in the reorganization lactobacillus rhamnosus
Switching reorganization lactobacillus rhamnosus is cultivated to the MRS that contains protoheme and concentrates, be cultured to logarithmic phase later stage results, get 5ml bacterium liquid, the centrifugal 5min of 12000r/min, supernatant discarded is with phosphoric acid buffer (0.05M, pH 7.8, contain 0.1mM EDTA) wash and be resuspended in the 2.5ml damping fluid, low temperature ultrasonication, dialysed overnight in phosphoric acid buffer afterwards.
Draw 500 μ l dialyzates, add 500 μ l ultrapure waters and 3ml 0.01% (W/W) Xylene Brilliant Cyanine G G-250 solution, react and measure the 595nm light absorption value after 3 minutes.
The bovine serum albumin standard model and 3ml 0.01% (W/W) the Xylene Brilliant Cyanine G G-250 solution that 1ml are contained different concns mix, and react and measure the 595nm light absorption value after 3 minutes, draw out light absorption value-protein content typical curve.
Utilize the protein content in the typical curve calculating dialyzate.
5.2 pyrogallol autoxidation method is measured the SOD enzyme and is lived
Get a test tube, add 100 μ l dialyzates successively, the 1.4ml ultrapure water, 150 μ l 6mM pyrogallol solution, 2.35ml 0.01M Tris-HCl (contain 2mM EDTA, pH 8.0), impouring cuvette behind the mixing is measured the variation of 420nm place light absorption value rapidly, does not add dialyzate as blank.Calculating dialyzate and suppress the autoxidizable per-cent of pyrogallol, is 1U to suppress 50% pyrogallol autoxidation, obtains the enzyme activity of SOD, unit: U/mg albumen in conjunction with the dialyzate protein content.
Through measuring, the SOD activity that changes the lactobacillus rhamnosus of plasmid pSIPCK and pSIPCAT over to is 0, and the SOD activity that changes the lactobacillus rhamnosus of cointegrating plasmid pSIPCS over to is 147.80U/mg albumen.Above-mentioned data all are the mean value that the test through 5 clones records.
6.1 reorganization lactobacillus rhamnosus tolerance H
2O
2Merit rating
Get the fresh logarithmic phase nutrient solution of 1ml, the centrifugal 5min of 12000r/min, supernatant discarded with the resuspended thalline of fresh substratum, adds 6mM H
2O
2, handle 1h for 37 ℃, (final concentration 10U/ml Sigma) digests remaining H to use beef liver catalase afterwards
2O
2, use the physiological saline gradient dilution, get suitable gradient coating, cultivate 24h and count single colony number later on; Not add H
2O
2The bacterium liquid of handling is reference, calculates survival rate.
Through measuring, the lactobacillus rhamnosus that changes pSIPCAT and pSIPCS over to is at 6mM H
2O
2The survival rate of handling behind the 1h is respectively 81.5% and 100%, exceeds nearly 1,000 times than the survival rate of the lactobacillus rhamnosus that contains control plasmid pSIPCK, and its anti-H is described
2O
2Ability is significantly improved; And change the H of the lactobacillus rhamnosus of cointegrating plasmid pSIPCS at greater concn
2O
2Survival rate after handling exceeds more than 100 times again than the lactobacillus rhamnosus that only contains CAT, illustrates that the acting in conjunction of CAT and SOD can further improve the anti-H of this lactobacillus rhamnosus
2O
2Toxic ability.
6.2 the growing state of reorganization lactobacillus rhamnosus under the aerated culture condition
The reorganization lactobacillus rhamnosus nutrient solution of getting 50 μ l incubated overnight is forwarded in the MRS substratum that 50ml contains protoheme, place shaking table to cultivate (37 ℃ 200rpm), are got 1ml bacterium liquid every 24h with the 250ml triangular flask, the coating counting is observed the growth tendency in 4 days behind the gradient dilution.
The results are shown in Figure 4, the viable count of the lactobacillus rhamnosus that contains CAT under long-term aerated culture condition approximately compared according to bacterial strain and exceeded 100 times, and its oxygen-resistant ability greatly improves.
The lactobacillus rhamnosus that carries pSIPCAT and pSIPCS of getting 50 μ l incubated overnight respectively is forwarded in the low sugar MRS substratum (glucose content is 0.125%) that 50ml contains protoheme, place shaking table to cultivate (37 ℃ with the 250ml triangular flask, 200rpm), every 24h coating counting, observe the growing state in the week.
The results are shown in Figure 5, cultivate after 5 days, the viable count that only contains the lactobacillus rhamnosus of CAT begins rapid decline, and the viable count that contains the dual-gene lactobacillus rhamnosus of CAT and SOD still keeps high level, and dual-gene acting in conjunction can further improve its oxygen-resistant ability really.Each experimental group all repeats 5 bacterial strains, and the testing data of gained is 5 clones' a mean value.
The sequence explanation:
SEQ ID NO1 is a lactobacillus sake katA nucleotide sequence; NO2 is a lactobacillus sake katA aminoacid sequence; NO3 is thermophilus streptococcus sodA and nucleotide sequence; NO4 is a thermophilus streptococcus sodA aminoacid sequence; NO5 and NO6 are right for the primer of amplification lactobacillus sake katA; NO7 and NO8 are right for the primer of amplification thermophilus streptococcus sodA.
Claims (7)
1. a recombinant vectors is characterized in that, carries bacterium catalase gene and superoxide dismutase gene.
2. recombinant vectors according to claim 1 is characterized in that described superoxide dismutase gene comprises self promotor.
3. recombinant vectors according to claim 1 is characterized in that, described catalase gene clone is from lactobacillus sake.
4. recombinant vectors according to claim 1 and 2 is characterized in that, described superoxide dismutase gene clone is from thermophilus streptococcus.
5. the lactobacillus rhamnosus engineering strain that contains each described recombinant vectors of claim 1～4.
6. the preparation method of the described reorganization of claim 5 lactobacillus rhamnosus engineering strain comprises the steps:
1) amplification catalase gene and the superoxide dismutase gene from the genome of lactobacillus sake and thermophilus streptococcus respectively of the method by PCR;
2) series connection of catalase gene that amplification is obtained and superoxide dismutase gene is building up on intestinal bacteria-Bacterium lacticum shuttle vector pSIP502, obtains recombinant plasmid;
3), cultivate intestinal bacteria and extract plasmid with the recombinant plasmid transformed intestinal bacteria;
4) by electroporation with recombinant plasmid transformed in lactobacillus rhamnosus.
7. the application of claim 5 described reorganization lactobacillus rhamnosus engineering strain in fermented food industry.
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| 《J Ind Microbiol Biotechnol》 20100921 Guohong Wang et al. Coexpression of bile salt hydrolase gene and catalase gene remarkably improves oxidative stress and bile salt resistance in Lactobacillus casei 全文 1-7 , 2 * |
| 《JORUNAL OF AGRICULTURAL AND FOOD CHEMISTRY》 20110318 Haoran An et al. Coexpression of the Superoxide Dismutase and the Catalase Provides Remarkable Oxidative Stress Resistance in Lactobacillus rhamnosus 3851-3856 1-7 第59卷, 2 * |
| 《Mol Biotechnol》 20100303 Haoran An et al. High-Level Expression of Heme-Dependent Catalase Gene katA from Lactobacillus Sakei Protects Lactobacillus Rhamnosus from Oxidative Stress 155-160 1-7 第45卷, 2 * |
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| CN108070547A (en) * | 2017-12-22 | 2018-05-25 | 淮阴师范学院 | Applied to anaerobic bacteria and the method for facultative anaerobic bacteria screening positive clone bacterial strain |
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