CN102559732A - Construction method of Sul-de-Quinone Reductase gene vector and genetic engineering lactic acid bacteria strain of Sul-de-Quinone Reductase gene vector - Google Patents

Abstract

The invention relates to a construction method of a Sul-de-Quinone Reductase gene vector (pMG36e-SQR-Myc) and a genetic engineering lactic acid bacteria strain of the Sul-de-Quinone Reductase gene vector and belongs to the technical field of organisms. The construction method comprises the following steps of: selecting pcDNA3.1-SQR-Myc eukaryotic expression plasmid as an initial raw material, connecting an SQR-Myc gene segment with a pMG36e vector and electrically-converting lactococcus lactis MG1363 and obtaining an efficient and stable expression. The construction method and the genetic engineering lactic acid bacteria strain have the beneficial effects that as selected lactic acid bacteria belongs to probiotics, the lactic acid bacteria can be taken as a safe micro-ecological preparation for directly feeding an animal; the lactic acid bacteria is habituated in an intestinal tract to achieve a normal physiological effect; and SQR lactic acid bacteria is expected to realize the function of degrading sulfide in the intestinal tract of the animal, so that the emission of sulfide in excrement is finally reduced.

Description

Sulfide quinone oxidoreductase genophore makes up and the transgenic engineering lactic bacterium strains

One, technical field

The present invention relates to sulfide quinone oxidoreductase genophore (pMG36e-SQR-Myc) and make up and the transgenic engineering lactic bacterium strains, belong to biological technical field.This commentaries on classics SQR genetic engineering bacterium can import in the animal and bird intestines settles down, and to reduce the generation and the discharging of sulfide waste gas in the enteron stool, reduces environmental pollution.

Two, background technology

Along with the fast development of herding industry, the continuous expansion of plant's scale, the waste gas (H that livestock and poultry (pig, chicken, ox etc.) ight soil produces ₂The S sulfides) become the air pollution source that can not be ignored, how making these waste gas innoxious is to guarantee one of prerequisite that environmentally friendly aquaculture develops in a healthy way.Yet, adopt ordinary methods such as stacking fermentation, biogas, absorption, burning and reodorant can reduce the pollution of waste gas to a certain extent at present to environment, can't accomplish innoxious.So it is very necessary to seek a kind of waste gas innoxious process for treating approach.

(Sul de-Quinone Reductase SQR) extensively is present in (genus chlorobium, bacillus capsulatus genus, Chromatium etc.) in phototrophy and the chemotrophy bacterium to sulfide-quinone oxidoreductase, and it is the polypeptide of a 57-kDa, can decomposing H ₂The S sulfides.1997, separation of pure from red bacillus capsulatus such as Sh ü tz dissolved SQR, and the SQR gene is successfully cloned in intestinal bacteria, and the result shows that the SQR of escherichia coli expression has bioenzyme activity.Over surplus in the of nearly ten year, scientist has carried out systematic research to the sequential structure of SQR gene and the biological function of degraded sulfide thereof both at home and abroad, obtains remarkable break-throughs, its special biological function utilize prospect first meeting clue.But do not carry out the relevant research that utilizes SQR to be used for to reduce the feces of livestock and poultry sulfide emission so far as yet.

Milk-acid bacteria is one of beneficial flora important in host's (humans and animals) enteron aisle, and the intestinal microecology balance is kept in adjusting, promote to take in nutrient digestion, absorb, keep host health state etc. and have the effect that is highly profitable.Successful separation along with the various expression regulation elements of milk-acid bacteria makes milk-acid bacteria become comparatively ideal transgenic engineering recipient bacterium.PMG36e is a kind of expressive plasmid carrier that is usually used in lactococcus lactis ssp, and this plasmid size is 36kb, contains erythromycin resistance gene, p32 promotor, MCS and prtp transcription terminator.Guchte and Brurberg etc. are as carrier exogenous genes products such as successful expression N,O-Diacetylmuramidase and chitinase in milk-acid bacteria.Zhao Ying etc. have successfully obtained acidic cellulase transgenic lactic bacilli strains; Zhao Dan etc. have then obtained to change the lactobacillus spp of chicken coccidia gene.At present, the research of transgenic lactobacillus spp is seen in fields such as Animal nutrition and animal diseases controls, and waste gas results from discharging in the application commentaries on classics SQR gene lactic bacteria reduction animal intestinal ight soil but be not involved in as yet, reduces the research of environment exhaust emission association area.

The SQR H that can degrade ₂The biological function of S sulfides has obtained certainly, but passes through the SQR degraded H that transgenic lactobacillus is expressed ₂This new way of S sulfides reduces the sulfide emission in the feces of livestock and poultry; Still need and solve all kinds of technical problem; As construct suitable SQR gene lactic bacteria expression plasmid; This plasmid through conversion be incorporated in the milk-acid bacteria genome and in the process that goes down to posterity genetic stability and SQR can in milk-acid bacteria, efficiently express etc., these sport technique segments must and be used in the research of carrying out SQR transgenic lactobacillus probiotics effectively being solved before the living animal experiment.To above-mentioned sport technique segment problem, the present invention constructs the milk-acid bacteria expression plasmid pMG36e-SQR of SQR gene, infects engineering lactic acid bacteria through electric method for transformation, identifies and filter out the lactic bacterium strains of high efficiency stable expression SQR, and through going down to posterity repeatedly, building is.For further carrying out the development of SQR transgenic lactobacillus probiotics, effectively solve H in the livestock and poultry cultivation process ₂A S sulfides environmental pollution difficult problem provides necessary experiment material and gordian technique to support.

Three, summary of the invention

Technical problem

The purpose of this invention is to provide and express SQR proteic recombinant vectors pMG36e-SQR construction process and transgenic lactobacillus engineering strain technology of preparing thereof, SQR can be stablized, expressed efficiently to the final transgenic lactobacillus strain that obtains.

Technical scheme

Express the proteic milk-acid bacteria of SQR, produce, comprising through following method:

(1) structure of milk-acid bacteria SQR protein expression vector pMG36e-SQR

1. pMG36e-SQR-Myc vector construction and evaluation thereof

It is initial starting material that the structure of SQR genophore pMG36e-SQR-Myc of the present invention is selected pcDNA3.1-SQR-Myc (Fig. 1) eukaryon expression plasmid for use; At first; Utilize restriction enzyme Kpn I and Xba I that the SQR goal gene is scaled off together with Myc marker gene (being SQR-Myc) enzyme from the pcDNA3.1-SQR-Myc carrier; Reclaim the SQR-Myc gene fragment; Adopt the T4 ligase enzyme to connect, be built into pMG36e-SQR-Myc (Fig. 3) prokaryotic expression carrier in the corresponding restriction enzyme site of pMG36e carrier (Fig. 2).Utilize the KCM conversion method to import in the bacillus coli DH 5 alpha transformant, containing screening on the LB substratum of Oxacyclotetradecane,erythromycin deriv, identifying positive transformant.

2. the recombinant plasmid that contains the SQR gene is identified

For confirming the positive of transformant, must carry out PCR method and the evaluation of double digestion method to recombinant plasmid pMG36e-SQR-Myc.Difference picking white colony on the solid medium of inoculating behind the KCM method transformed into escherichia coli; Be seeded in the LB substratum and cultivate; Alkaline lysis extracts plasmid on a small quantity, identifies that through double digestion goal gene SQR and marker gene Myc are connected in the corresponding site (see figure 4) of pMG36e.With the plasmid is template, and P1, P2 are that primer increases, and identify positive recombinant plasmid.Primer P1 TCGTCACCAAGGACCCGATGTAT, P2 TCACGGCCTTCAGCTTGTCG, reaction conditions are 94 ℃; Preparatory sex change 5min, 94 ℃ of sex change 10s, 51 ℃ of annealing 30s; 72 ℃ are extended 80s, totally 30 circulations, and 72 ℃ are extended 10min; Amplified production carries out agarose gel electrophoresis, detects this recombinant plasmid and contains SQR gene (Fig. 5).

(2) electricity of lactococcus lactis ssp MG1363 transforms

1. the competent preparation of lactococcus lactis ssp MG1363

MG1363 is in the GM17 substratum in inoculation; 30 ℃ of overnight cultures; Being inoculated in the height that contains 1% glycocoll by 5% inoculum size, to ooze GM17 substratum (SGM17) to OD value be 0.2 ~ 0.8, cultivate finish after with bacterial cultures ice bath 10min, centrifugal collection thalline; Ice-cold Phosphoric acid glycerol esters damping fluid washing thalline 3 times adopts the 1/100 long-pending resuspended thalline of electric shock damping fluid of bacteria liquid at last.

2. electroporation transforms MG1363

Plasmid pMG36e-SQR-Myc mixes with above-mentioned competence MG1363, ice bath 10min, electric shock.Electricity commentaries on classics parameter is following: voltage 2000 V, electric capacity 25 μ F, resistance 400 Ω.After 3 h are cultivated in 30 ℃ of recoveries, it is coated on the Oxacyclotetradecane,erythromycin deriv GM17 flat board that contains 10 μ g/ mL, cultivates 2～3 d, obtain the positive transformant bacterium colony.

(3) expression of SQR in lactococcus lactis ssp MG1363

Get positive lactic bacterium strains, be inoculated in the 5mL liquid GM17 substratum incubated overnight; Collect thalline, wash 2 times, add final concentration 10mg/mL N,O-Diacetylmuramidase 100 μ l with distilled water; 37 ℃ digest 30min, add the sample-loading buffer of equivalent, 100 ℃ of effect 5 min; Be used for the SDS-PAGE electrophoresis behind the high speed centrifugation, the result shows the band (Fig. 6) that contains the target protein size in the expression product.

Beneficial effect

The present invention has successfully made up the lactic acid bacteria expression vectors pMG36e-SQR-Myc of SQR; Expression for testing goal Protein S QR; Simultaneously label protein Myc also is connected into wherein, and through whether successful connection of two kinds of methods evaluations of double digestion and PCR pMG36e-SQR-Myc.Obtain competent lactococcus lactis ssp MG1363; The step of going forward side by side joint is optimized electricity and is transformed parameter (voltage, electric capacity etc.); Set up the efficient electricity that transforms lactococcus lactis ssp and transform the parameter condition; Obtain to express the lactococcal strain of SQR, the expression of SQR detects through SDS-PAGE electrophoresis and Western Blot.

Have the bibliographical information of escherichia coli expression SQR up to now, but do not see the expression study of SQR in milk-acid bacteria as yet.Why opt lactic acid bacteria is because milk-acid bacteria is the probiotic bacterium class; Can be used as the direct feeding animals of a kind of safe probiotics; Make it the perch physiological action of in enteron aisle, bringing into normal play; And the SQR milk-acid bacteria is expected to the function of performance degraded sulfide in animal intestinal, finally reduces the discharging of sulfide in the ight soil.

Four, description of drawings

Fig. 1: pcDNA3.1 (-)-SQR-Myc recombinant plasmid synoptic diagram.

Fig. 2: pMG36e plasmid map synoptic diagram, the blue line mark is respectively Xba I and Kpn I restriction enzyme site.

Fig. 3: pMG36e-SQR-Myc reconstruct plasmid synoptic diagram, the on position of demonstration SQR-Myc.

Electrophorogram after Fig. 4: pMG36e-SQR-Myc process Kpn I and Xba I enzyme are cut.

M:Marker; 1 cuts the goal gene band (2 repetitions) that the back produces with the 2:pMG36e-SQR-Myc enzyme.

Fig. 5: the pMG36e-SQR-Myc pcr amplification is identified figure.

M:Marker; 1-4:pMG36e-SQR-Myc pcr amplification product band (4 repetitions).

Fig. 6: the SDS-PAGE collection of illustrative plates of expressing SQR in the transgenic lactococcus lactis ssp.

M: albumen Marker; 1: negative control; 2,3:pMG36e-SQR-Myc-MG1363 positive strain, arrow labeled are the target protein band.

The expression Western check and analysis of Fig. 7: SQR in milk-acid bacteria.

M: albumen Marker; 1: negative control; 2, the total protein that extracts in the 3:pMG36e-SQR-Myc-MG1363 positive strain, arrow indication are the SQR zymoprotein band of destination gene expression.

Fig. 8: the growth curve chart that changes SQR lactococcus lactis ssp MG1363.

Five, embodiment

(1) structure of milk-acid bacteria SQR protein expression vector pMG36e-SQR-Myc (Fig. 1-5)

1. main experiment material

(2011,27 (5): 1043-1046), pMG36e buys by winning profit biotech company plasmid: pcDNA3.1-SQR-Myc for Yu Fengxiang etc., Jiangsu agricultural journal.

Bacterial classification: bacillus coli DH 5 alpha is purchased the precious biological ltd in Dalian.

Enzyme and reagent: the damping fluid of restriction enzyme Kpn I and Xba I, T4 ligase enzyme, endonuclease reaction, DL5000 Marker, PCR premix are all available from Dalian Takara company; Glue reclaims test kit, plasmid extracts test kit in a small amount available from sky root biochemical technology company; All the other chemical reagent are homemade, import packing or import.

2. solution preparation

The preparation of substratum, reagent all is solvent with the redistilled water if having no special requirements, and the autoclaving condition is 102.9kPa vapor sterilization 30 minutes.

The LB liquid nutrient medium:Peptone l0g, yeast powder 5g, NaCl l0g is dissolved in the 800m1 water, regulates pH value to 7. 5, is settled to 1000m1, autoclaving.

The LB solid medium:Peptone 10g, yeast powder 5g, NaCl l0g is dissolved in the 800m1 water, adds agar powder 15g, regulates pH value to 7. 5, is settled to 1000m1, autoclaving.

The Oxacyclotetradecane,erythromycin deriv preparation:Oxacyclotetradecane,erythromycin deriv is dissolved in the absolute ethyl alcohol, and dense stock concentrations is 50mg/ml, working concentration 250 μ g/ml.

The preparation of KCM conversion method related reagent:

(1) TSM liquid preparation (10ml)

Mother liquor	Volume	Final concentration
			LB	7.3m1
50% PEG MW4000	2 ml	10%
			DMSO	0.5ml	5%
1M MgC12	100μl	l 0mM
			1M MgS04	100μl	l 0mM

(2) 5 * KCM:0.5M KCl, 0.15M CaC1 ₂, 0.25M MgC1 ₂After the filtration sterilization, divide to be filled in the 1.5m1 EP pipe, frozen subsequent use in one 20 ℃ of refrigerators.

Plasmid extracts solution I:50mmol/L glucose, 25mmol/L TrisCl (pH8.0), l0mmol/L EDTA (pH8. 0), autoclaving 15 minutes.

Plasmid extracts solution II:0. 2mmol/L NaOH, 1% SDS, existing with join at present.

Plasmid extracts solution III:5mol/L potassium acetate 60m1, glacial acetic acid 11. 5m1, water 28. 5m1,4 ℃ of preservations.

50 * TAE:242g Tris alkali, the 57.1ml glacial acetic acid, 10,0m1 0. 5mo1/L EDTA (pH 8.0) is settled to 1000m1 after the dissolving.

3. experimental procedure

(1) endonuclease reaction system

pcDNA3.1-SQR-Myc 50ng pMG36e 50ng

Kpn I 1μl Kpn I 1μl

Xba I 1μl Xba I 1μl

10×buffer 2μl 10×buffer 2μl

Moisturizing to 20 μ l, 37 ℃ of water-bath 3 h, 1% agarose electrophoresis also reclaims the SQR-Myc target gene fragment and the pMG36e carrier segments.

(2) agarose gel reclaimer operation reference reagent box specification sheets carries out.

(3) ligation

Calculate the amount of exogenous segment and carrier: size (the kb) * insertion fragment of size (kb)/carrier of insertion DNA (ng)=carrier (ng) * insertion DNA and the molecular ratio of carrier.The general sheet segment molecule that inserts: carrier molecule=3 ~ 8: 1

Ligation system (10 μ 1):

The 20ng carrier DNA

The 20ng foreign DNA

1 μ, 1 T4DNA ligase enzyme, 10 X buffer

1 ~ 2U T4 dna ligase

Benefit adds water to 1,16 ℃ of incubation 8 ~ 12h of 10 μ.Get 5 μ 1 and connect liquid conversion, all the other-20 ℃ of preservations.

(4) KCM method transformed into escherichia coli

The competent preparation of bacillus coli DH 5 alpha: prepare and from frozen bacterial classification pipe, chose bacterium in preceding 1 day to 4m1 LB pipe, 37 ℃ are shaken bacterium and spend the night; 4m1 bacterium liquid is transferred to 100m1 antibiotic-free LB substratum, and 37 ℃ are shaken about the about 1h of bacterium till OD600=0.3 ~ 0.4; 100m1 bacterium liquid is transferred in the 50mI centrifuge tube, in 4 ℃ centrifugal, remove supernatant, under condition of ice bath, blow and beat and be resuspended in the 10 ml TSB liquid, put 10 minutes on ice; Adding 50% glycerine to glycerine final concentration is 10%, the piping and druming mixing; Divide in the 0.5 mL centrifuge tube that is filled to precooling (every pipe is adorned 55 μ l), place the liquid nitrogen quick-frozen immediately, be put in-70 ℃ of cryogenic refrigerators afterwards and preserve.

Connect the conversion of product: prepare in 1 * DNA mixture, i.e. 5 * KCM, 10 μ l, water 30 μ l connect product 10 μ l, and the rearmounted 65 ℃ of water-baths of totally 50 μ l piping and druming mixing are 10 minutes; (55 μ l) is added in the said mixture with competence bacteria liquid, rotates the mixing content gently; In ice, placed 20 minutes, took out rearmounted room temperature 10 minutes; Add in LB liquid culture to the above-mentioned competent cell/DNA mixture of 400 μ l antibiotic-frees, put 37 ℃ of shaking tables and shake bacterium at a slow speed more than 30 minutes; Bacterium liquid is centrifugal, outwell supernatant, resuspended deposition, bacterium liquid all transferred to contain 250 μ g/ml Oxacyclotetradecane,erythromycin deriv the LB flat board with the screening positive transformant; Be inverted flat board in 37 ℃, incubated overnight 12 ~ 16h.

(5) evaluation of transformant

Restriction enzyme digestion and electrophoresis is identified: select white colony and be inoculated in the LB substratum; Alkaline lysis extracts plasmid on a small quantity (referring to " molecular cloning experiment guide "; Volumes such as second edition [U.S.] J Sa nurse Brooker; The related Sections that Quan Dongyan etc. translate), the endonuclease reaction system is plasmid 5 μ l, Kpn I and Xba I each 0.5 μ l, 10 * buffer, 2 μ l, supplies water 20 μ l, electrophoresis evaluation on 1% agarose gel behind 37 ℃ of reaction 2h.

PCR identifies: same picking white colony, and be seeded in the LB substratum and cultivate, alkaline lysis extracts plasmid on a small quantity, is template with the plasmid; P1, P2 are that primer increases, primer P1 TCGTCACCAAGGACCCGATGTAT, and P2 TCACGG CCTTCAGCTTGTCG, reaction conditions are 94 ℃; Preparatory sex change 5min, 94 ℃ of sex change 10s, 51 ℃ of annealing 30s, 72 ℃ are extended 80s; Totally 30 circulations, 72 ℃ are extended 10min, and amplified production carries out agarose gel electrophoresis.

4. result

Through above experimental procedure; We have successfully made up the lactic acid bacteria expression vectors pMG36e-SQR-Myc (Fig. 3) of SQR; And adopting double digestion method enzyme to cut out goal gene SQR fragment, size is 1389bp (Fig. 4), adopts PCR method to amplify goal gene SQR fragment simultaneously; Size is 1179bp (Fig. 5), explains successfully SQR-Myc to be connected among the lactic acid bacteria vector pMG36e.

(2) detection of expression (Fig. 6,7) of SQR in lactococcus lactis ssp MG1363

1. material

Bacterial classification: lactococcus lactis ssp MG1363 is so kind as to give (Lv Xiaoying etc., Chinese microecology magazine, 2001,13 (5): 265-266) by Kaifeng, Henan Province chemical engineering institute of university Chen Jin peak.

Reagent: the mouse one goat-anti mouse two anti-, horseradish peroxidase of anti-Myc resists, Pro-light HRP chemiluminescence detection reagent is all purchased the root biochemical corp in the sky.SDS-PAGE and Western Blot related reagent are given birth to the worker available from Shanghai, and developing solution, stop bath are available from the just right scientific & technical corporation in Nanjing.

2. solution preparation

The preparation of substratum, reagent all is solvent with the redistilled water if having no special requirements, and the autoclaving condition is 102.9kPa vapor sterilization 30 minutes.

GM17 substratum (1L): Tryptones 5g, soy peptone 5g, yeast extract 2.5g, Carnis Bovis seu Bubali cream 5g, glycerine 10g, Sodium phosphate, dibasic 10g, Vc 0.5g, sal epsom 0.25g, glucose 5g, lactose 5g.

Height oozes substratum (SGM17): GM17+10% sucrose (Holo H & Nes IF, Appl Environ Microbiol, 1989,55 (12): 3119-3123).

Phosphoric acid glycerol esters damping fluid: 10% glycerine, 0.3M sucrose, 80mM MgCl ₂, 5 mM Na ₂HPO ₄-NaH ₂PO ₄(pH 7.4).

Electricity changes damping fluid: 0.3M sucrose+10% glycerine+1mM phosphoric acid buffer (Na ₂HPO ₄-NaH ₂PO ₄, pH 7.4)+0.1mM MgCl ₂

Recovery media (SGM17MC): add 20mM MgCl among the SGM17 ₂With 2mM CaCl ₂

SDS-PAGE and the preparation of WESTERN BLOT related solution:

(1) 30% acrylic amide: acrylic amide 29g, methylene diacrylamide 1g, adding distil water filter in the rearmounted brown bottle to 100ml.4 ℃ keep in Dark Place.(2) 1.5 M pH8.8 Tris-HCl damping fluids: take by weighing Tris 18.15g, add 50ml water, transfer pH 8.8 with 1mol/L hydrochloric acid, zero(ppm) water is settled to 100ml.(3) 0.5 M pH6.8 Tris-HCl damping fluids: take by weighing Tris 6.05g, add 50ml water, transfer pH 6.8 with 1mol/L hydrochloric acid, zero(ppm) water is settled to 100ml.(4) 10% ammonium persulphates: take by weighing the 1g ammonium persulphate, add 10ml water.

(5) SDS-PAGE sample loading buffer: pH 6.8 0.5M Tris damping fluid 2.5ml, glycerine 2ml, 10%SDS 2ml, mercaptoethanol 0.2ml, 0.1% tetrabromophenol sulfonphthalein 0.4ml, H ₂O 2.9ml mixing.

(6) 10 times of electrode buffers: Tris 30g, glycocoll 144g adds SDS 10g, and adding distil water is settled to 1000ml after making its dissolving.

(7) Xylene Brilliant Cyanine G dye liquor: take by weighing coomassie brilliant blue R250 1g and add 50ml methyl alcohol, the 10ml Glacial acetic acid min. 99.5 adds water to 100ml

(8) Xylene Brilliant Cyanine G gel destainer: 150ml methyl alcohol, 100 ml Glacial acetic acid min. 99.5 add water to 1000ml

(9) transfering buffering liquid (1L): take by weighing 3g glycocoll, 14.4gTris alkali, and add 200m l methyl alcohol, add water to total amount 1L.

（10）TBST ：20 mM Tris-HCl（PH7.5）, 150 mM NaCl，0.05%（V/V）Tween 20

Confining liquid (5% skim-milk): skim-milk 5g is dissolved in 100 ml TBST

(11) 0.5% ponceau dye liquors: Ponceau S 0.5g is dissolved in the ethanol of 100 ml 1%.

(12) developing solution, stop bath are prepared to specifications.

3. experimental procedure

(1) the competent preparation of lactococcus lactis ssp MG1363

MG1363 is inoculated in the GM17 substratum; 30 ℃ of overnight cultures; Being inoculated in by 5% inoculum size and being cultured to the OD value among the SGM17 that contains 1% glycocoll is 0.2 ~ 0.8, ice bath 10min, centrifugal collection thalline; Ice-cold Phosphoric acid glycerol esters damping fluid washing thalline 3 times adopts the 1/100 long-pending resuspended thalline of electric shock damping fluid of bacteria liquid at last.

(2) electroporation transforms MG1363

The plasmid pMG36e-SQR-Myc of 0.5-1 μ g is mixed ice bath 10min, electric shock with 40 μ l competence MG1363.Shock parameters is following: voltage 2000 V, electric capacity 25 μ F, resistance 400 Ω.After 3 h are cultivated in 30 ℃ of recoveries, it is coated on the Oxacyclotetradecane,erythromycin deriv GM17 flat board that contains 10 μ g/ mL, cultivates 2～3 d.

(3) SDS-PAGE detects the expression of SQR at MG1363

Picking is positive MG1363Bacterial strain is inoculated in the 5mL liquid GM17 substratum, and incubated overnight is collected thalline; Distilled water is washed 2 times, adds final concentration 10 mg/mL N,O-Diacetylmuramidases 100 μ l, 37 ℃ of digestion 30min; The sample-loading buffer that adds equivalent, 100 ℃ of effect 5 min carry out the SDS-PAGE electrophoresis behind the high speed centrifugation.

(4) Western blot detects the expression of SQR at MG1363

After the SDS-PAGE electrophoresis finishes, take out gel, pvdf membrane soaks 15s in methyl alcohol; Soak 2 min in the distilled water; Transfer to then in the transfering buffering liquid and to soak 5 min, the structure of filling up by sponge pad-filter paper-gel-film-filter paper-silk floss successively with pvdf membrane is subsequently packed into during transfer printing presss from both sides, and connects transfer device and puts it in the transfering buffering liquid of ice; 300mA, 90 min.After changeing film and finishing, pvdf membrane is put in 5% skim-milk seals, room temperature is shaken 2 h, and anti-Myc mouse one anti-1:1000 dilution is spent the night; TBST washs 10 min, washes altogether 3 times, and two anti-1:500 dilutions, room temperature is shaken 2 h; TBST washs 10 min, washes altogether 3 times, and isopyknic A liquid and B liquid mixing are promptly got in the ECL colour developing; Be mixed with the substrate working fluid, take out pvdf membrane, lie on the clean preservative film, the facing up of film; Central authorities at film add an amount of substrate working fluid that configures, and pvdf membrane is wrapped and put into magazine with preservative film, treat that putting into the X-mating plate according to the signal power after signal occurs makes public, and develop and photographic fixing to the X-mating plate in the exposure back.

4. result

Through above experimental procedure, we successfully utilize electric method for transformation to import in the lactococcus lactis ssp recombinant plasmid pMG36e-SQR-Myc, and adopt SDS-PAGE electrophoresis (Fig. 6) and Western blot to detect (Fig. 7) to the expression of SQR in MG1363.

(3) genetic stability of pMG36e-SQR-Myc in lactococcus lactis ssp MG1363 detects (Fig. 8, table 1)

1. the mensuration of growthing lag phase and generation time

The MG1363 that picking changes SQR is inoculated in the GM17 substratum, and 37 ℃ of incubated overnight are got 1ml bacterium liquid mixing in 100ml GM17 substratum, be sub-packed in the 10 ml test tubes, and each test tube 2ml, 37 ℃ leave standstill cultivation.Each h gets 3 test tubes and surveys bacterium liquid OD value, and with bacterium liquid dilution certain multiple, is inoculated in the GM17 flat board, cultivates 24h for 37 ℃, calculates viable count/ml.With the incubation time is X-coordinate, the bacterium liquid OD value of surveying be that ordinate zou draws growth curve chart (Fig. 8).The growth wherein one section of from growth curve, taking the logarithm is calculated generation time by following formula, G=(t1-t0) ln2/ (lnx-lnx0), and wherein G is generation time; T1 is an incubation time; The t0 initial incubation time, the viable count when x0 is beginning, x is the viable count after the t time cultivates.According to growth curve chart that Fig. 8 drew and calculate the formula of generation time, can calculate under this laboratory condition, the MG1363 growth lag phase that changes SQR is 2 h, is about 43 min generation time.

2. the genetic stability of plasmid detects among the SQR transgenic MG1363

Utilize the characteristic that contains erythromycin resistance gene in the pMG36e-SQR-Myc plasmid, its engineering bacteria is being contained Oxacyclotetradecane,erythromycin deriv and do not containing in the liquid GM17 substratum of Oxacyclotetradecane,erythromycin deriv continuous passage to 120 generation, whenever make plasmid stability mensuration one time at a distance from 10 generations.Choose 100 single bacterium colony * 100% on the flat board of the colony count that plasmid index of stability=contain grows on the flat board of Oxacyclotetradecane,erythromycin deriv at last/do not contain Oxacyclotetradecane,erythromycin deriv, detailed process is following:

From former generation bacterial strain plate picking list bacterium colony in the liquid nutrient medium of the GM17 that contains 10ug/ml Oxacyclotetradecane,erythromycin deriv; Overnight cultures; Be inoculated in the liquid nutrient medium of the GM17 that contains Oxacyclotetradecane,erythromycin deriv and do not contain Oxacyclotetradecane,erythromycin deriv 37 ℃ in second day by 1% inoculum size and leave standstill cultivation; Every at a distance from 10 generations (9h), tube obtains the bacterium liquid of different algebraically.The bacterium liquid of getting for 10-120 generations respectively by the certain multiple dilution after, coat the flat board of the GM17 that does not contain Oxacyclotetradecane,erythromycin deriv, cultivate 24h for 37 ℃.100 dibblings of picking list bacterium colony are in the corresponding flat board that contains or do not contain Oxacyclotetradecane,erythromycin deriv respectively, 37 ℃ of overnight cultures, and computational engineering bacterium MG1363 is at the Loss Rate that has or not the plasmid that goes down to posterity under the erythromycin selection pressure.

Obtain data shown in the table 1 according to above-mentioned experimental implementation, the result shows that the plasmid genetic stability among the SQR transgenic MG1363 that is obtained is better, can reach more than 95%, explains that recombinant plasmid wherein is difficult for losing.

Table 1 changes the genetic stability of plasmid among the SQR lactococcus lactis ssp MG1363

SEQUENCE LISTING

< 110>Jiangsu Province Agriculture Science Institute

< 120>sulfide quinone oxidoreductase genophore makes up and the transgenic engineering lactic bacterium strains

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Claims (3)

1. sulfide quinone oxidoreductase genophore pMG36e-SQR-Myc, its construction process comprises:

1) pMG36e-SQR-Myc vector construction

Selecting the pcDNA3.1-SQR-Myc eukaryon expression plasmid for use is initial starting material; Utilize restriction enzyme Kpn I and Xba I that the SQR goal gene is scaled off together with Myc marker gene SQR-Myc enzyme from the pcDNA3.1-SQR-Myc carrier; Reclaim the SQR-Myc gene fragment; Adopt the T4 ligase enzyme to connect, be built into the pMG36e-SQR-Myc prokaryotic expression carrier in the corresponding restriction enzyme site of pMG36e carrier; Utilize the KCM conversion method to import in the bacillus coli DH 5 alpha, containing screening on the LB substratum of Oxacyclotetradecane,erythromycin deriv, identifying positive transformant;

2) contain the recombinant plasmid positive identification of SQR gene

Picking positive transformant white colony carries out PCR method and the evaluation of double digestion method respectively:

Be seeded in the LB substratum and cultivate, alkaline lysis extracts plasmid on a small quantity, identifies that through double digestion goal gene SQR and marker gene Myc are connected in the corresponding site of pMG36e;

With the plasmid is template, according to SQR gene order design primer P1, P2, identifies positive recombinant plasmid, primer P1 TCGTCACCAAGGACCCGATGTAT; P2 TCACGGCCTTCAGCTTGTCG, reaction conditions are 94 ℃, preparatory sex change 5min, 94 ℃ of sex change 10s; 51 ℃ of annealing 30s, 72 ℃ are extended 80s, totally 30 circulations, 72 ℃ are extended 10min; Amplified production carries out agarose gel electrophoresis, contains the band of SQR gene, and size is 1179bp.

2. transform the transgenic engineering lactic bacterium strains that obtains with the said carrier of claim 1, obtain by following steps:

1) the competent preparation of lactococcus lactis ssp MG1363

MG1363 is in the GM17 substratum in inoculation; 30 ℃ of overnight cultures; To be inoculated in SGM17 substratum to the OD value of the glycocoll that contains volume ratio 1% be 0.2 ~ 0.8 to 5% inoculum size by volume, cultivate finish after with bacterial cultures ice bath 10min, centrifugal collection thalline; Ice-cold Phosphoric acid glycerol esters damping fluid washing thalline 3 times adopts the 1/100 long-pending resuspended thalline of electric shock damping fluid of bacteria liquid at last;

2) electroporation transforms MG1363

Plasmid pMG36e-SQR-Myc mixes with above-mentioned competence MG1363, ice bath 10min, and electric shock, electricity commentaries on classics parameter is following: voltage 2000 V, electric capacity 25 μ F, resistance 400 Ω,

After 3 h are cultivated in 30 ℃ of recoveries, it is coated on the Oxacyclotetradecane,erythromycin deriv GM17 flat board that contains 10 μ g/ mL, cultivates 2～3 d, obtain transgenic engineering lactic bacterium strains bacterium colony.

3. the said transgenic engineering lactic bacterium strains of the claim 2 sulfide quinone oxidoreductase of expressing, obtained by following steps: the weighting profit requires 2 said transgenic engineering lactic bacterium strains, is inoculated in the 5mL liquid GM17 substratum; Incubated overnight is collected thalline, washes 2 times with distilled water; Add final concentration 10mg/mL N,O-Diacetylmuramidase 100 μ l, 37 ℃ digest 30min, add the sample-loading buffer of equivalent; 100 ℃ of effect 5 min are used for the SDS-PAGE electrophoresis behind the high speed centrifugation, the result shows the band that contains target protein in the expression product; Size is 53KD, is the sulfide quinone oxidoreductase of the said transgenic engineering lactic bacterium strains expression of acquisition.

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