CN1624139A

CN1624139A - Process for expressing gene of superoxide mutase and its special carrier

Info

Publication number: CN1624139A
Application number: CN 200410091118
Authority: CN
Inventors: 王�琦; 王黎明; 梅汝鸿
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2004-11-18
Filing date: 2004-11-18
Publication date: 2005-06-08

Abstract

A special carrier for SOD gene expression is prepared by inserting SOD gene to the polyclonal site of Pichia yeast expression carrier. A method for expressing the SOD gene includes such steps as configuring the Pichia yeast expression carrier containing SOD gene, introducing it to Pichia yeast to obtain recombinant Pichia yeast and inducing the expression of SOD gene.

Description

A kind of expression method of superoxide dismutase gene and dedicated carrier thereof

Technical field

The present invention relates to method and dedicated carrier thereof that superoxide dismutase gene is expressed.

Background technology

Superoxide-dismutase (superoxide dismutase is extensively to be present in biological intravital metalloenzyme SOD), and it has broad variety, as: Fe-SOD, Mn-SOD and Cu/Zn-SOD etc.The SOD enzyme is unique a kind of with ultra-oxygen anion free radical (O in the body ^- ₂) be the enzyme of substrate, it can single-minded ground catalysis O ^- ₂Disproportionation reaction takes place, and generates H ₂O ₂And O ₂, thereby can resist the toxicity of oxyradical and other oxide compound radical pair cytoplasmic membrane preferably, the oxidative damage of defence body is played crucial effect (Zhao Baolu.Oxyradical and natural antioxidants.Science Press, 1999,3-125).SOD is mainly used in multiple diseases such as treatment oxygen intoxication, senile cataract, diabetes, cardiovascular disorder, various inflammation clinically; SOD also can be used as antiradiation agent, is used for the protection and the transplanting of organs such as auxiliary radiotherapy and chemotherapy and kidney, liver, heart, to reduce the side effect of high dose radiation.SOD is used to produce chewing gum, beverage and beer (Fang Yunzhong, Li Wenjie as foodstuff additive.Free radical and enzyme.Science Press, 1989).In addition, SOD is added in the makeup, also can play the effect that sun-proof, anti-skin aging and lipofuscin form.

At present, the preparation of SOD source mainly contains animal, plant and microorganism.SOD product major part is in the market extracted from animal blood.Reasons such as, purification difficult limited owing to starting material, cause the purity of SOD low, output is few, particularly along with crow epidemic disease and report frequently of mad cow disease all over the world, bird flu, mouth by pernicious transmissible diseases such as synzoic SARS, the risk of production animal source blood products strengthens, in addition, increasing of product purity requirement also increased production cost.Therefore, searching is cheap, heterologous gene expression system is one of effective way that breaks through the traditional preparation process method efficiently.

The methanol yeast expression system is nearly ten years eukaryotic expression systems that grow up, and compares with traditional escherichia expression system and first-generation yeast expression system one yeast saccharomyces cerevisiae, and methanol yeast has incomparable advantage.It not only has, and the prokaryotic organism growth is quick, substratum is cheap and the characteristics of technical scheme simple possible, has possessed typical Eukaryotic molecular biological characteristic simultaneously again, as processing folding and posttranslational modification etc. to expressed proteins; In addition, it has also remedied some defectives of prokaryotic expression system, exist with the inclusion body form as expression product is many, and the renaturation difficulty, expression efficiency low (＜20%), foreign protein is many, purification difficult etc.

Pichia pastoris phaff is a kind of in the methanol yeast, up to now, more than 400 kind of foreign protein successful expression (Cereghino, J L.and cregg J M.Heterologous proteinexpression in the methylotrophic yeast Pichia pastoris.FEMS micrrobiol. in this system have been reported

Rev.2001，24，45-66)。Compare with yeast saccharomyces cerevisiae, pichia spp has stronger promotor, thereby secernment efficiency is higher, expression plasmid is more stable, and can be used for high density fermentation (dry cell weight＞100g/L) (Romanos MA, Scorer CA, Clare J J.Foreign gene expression in yeast.Yeast, 1992,8:423-488).It can be grown in the substratum that with methyl alcohol is the sole carbon source and the energy, this is because contain the necessary enzyme of methanol metabolic pathway in the peroxysome of its cell, as alcohol oxidase, Protosol synthetic enzyme and catalase etc., wherein alcohol oxidase (AOX) is that methyl alcohol utilizes first enzyme in the approach, and its catalysis methanol is oxidized to formaldehyde and hydrogen peroxide.AOX is by AOX1 and two genes encodings of AOX2, and the AOX2 gene is similar to the AOX1 gene order, and 92% homology is arranged, and its proteins encoded has 97% homology; The strictness of AOX1 gene is subjected to methanol induction and regulation and control, and the coded product of AOX1 gene plays a major role in oxidising process.In the methyl alcohol cultured cells, alcohol oxidase can account for more than 30% of total protein, and with glucose, glycerine, when ethanol is carbon source for growth, then can not detect the activity of AOX.Alcohol oxidase gene (AOX1) promotor that pichia yeast expression system uses is strong inducible promoter, can regulate and control efficiently expressing of foreign gene effectively; Adopt integrating vector, can be to yeast chromosomal with exogenous origin gene integrator, thus obtain the bacterial strain of inheritance stability; The expression amount height, there are many proteic expression levels can reach more than every gram liter, as tetanus toxin C fragment expression amount up to 12g/L (Clare JJ, Rayment FB, Ballantine SP, et al.High-lever expession of tananus toxin fragment C in pichia pastorisstrains containing multiple tandem integration of the gene.Bio Technology, 1991,9:455-460.), the gelatin expression amount reaches 14.8g/L (Mare WT, et al.High-yieldsecretion of recombinant gelations by Pichia pastoris.Yeast, 1999,15:1087-1096).

Bacillus cereus M22 (Bacillus.cereus M22) is a kind of endophytic bacterium, be rich in the SOD enzyme in its body, the SOD that obtains in this thalline is carried out physical and chemical property determining to be found, its stability is higher than isolating SOD (Suning from plant, animal, clothing Wen Ping, Wang Qi etc., research (the Chinese microecology magazine of wax bacillus superoxide-dismutase physico-chemical property.1999，11(1)：7-9)。

Summary of the invention

The purpose of this invention is to provide method and dedicated carrier thereof that a kind of superoxide dismutase gene is expressed.

The dedicated carrier of expression superoxide dismutase gene provided by the present invention is the yeast expression vector that contains superoxide dismutase gene, AOX1 promotor and terminator sequence, multiple clone site and screening sign.

Described superoxide dismutase gene is between EcoR I of the multiple clone site place of yeast expression vector and Kpn I restriction enzyme digestion sites.

Described screening is masked as the complementation screening sign or the Zeocin of group ammonia alcohol dehydrogenase gene (HIS4) ^RScreening sign, wherein Zeocin ^RThe screening sign is preferred.

Described superoxide dismutase gene can be any one superoxide dismutase gene, preferably Mn-SOD and CuZn-SOD gene.

PICZ α A is that most preferred pichia spp born of the same parents express carrier outward, because of it possesses characteristics such as expression level height, screening and purifying be quick, and contain AOX1 promotor and terminator sequence and Zeocin resistant gene, can directly screen, improved screening efficiency transformant; In addition, in its multiple clone site downstream, also merging has 6 * His label and Myc site, helps the purifying and the detection of target protein.The physical map of PICZ α A plasmid as shown in Figure 1.

With PICZ α A is that the set out yeast expression vector that contains the Mn-SOD gene of vector construction is pPICZ α A-sodM18; The yeast expression vector that contains superoxide-dismutase base CuZn-SOD gene is pPICZ α A-sodC.

The method that superoxide dismutase gene provided by the present invention is expressed, be to make up the yeast expression vector that contains superoxide dismutase gene, the yeast expression vector that makes up is imported in the pichia spp, obtain recombinant yeast pichia pastoris, induce recombinant yeast pichia pastoris to make superoxide dismutase gene obtain expressing.

For improving transformation efficiency, in the importing pichia spp is linearizing yeast expression vector; The method that the restructured Pichia pastoris in expression carrier is imported in the pichia spp is preferably the sharp conversion method of electricity, but also can adopt method commonly used in other bioengineering field, for example lithium chloride conversion method, protoplast transformation method.

Need add methyl alcohol when inducing recombinant yeast pichia pastoris, add methyl alcohol concentration be 0.4-0.6%.For the volatilization loss of compensation methyl alcohol, also to add methyl alcohol in per 24 hours during fermentation, O.4-0.6% the methanol concentration in the bacterium liquid is remained on.The time of inducing restructured Pichia pastoris in expression is 2-6 days.

The method that superoxide dismutase gene provided by the present invention is expressed in pichia spp has remedied the deficiency of traditional SOD preparation method and prokaryotic expression system phraseology; In addition, if apply the present invention to the production of industrialization SOD, then have raw material biology growing cycle weak point, industrial scale is big, and the advantage that extraction cost is low has important prospects for commercial application and practical significance.

Description of drawings

Fig. 1 is the physical map of pichia spp secreted expression carrier pPICZ α A.

Fig. 2 is the vector construction figure that contains superoxide dismutase gene.

Fig. 2 a contains the design of graphics of the carrier pET-22b-sod of superoxide dismutase gene.

Fig. 3 is Mn-sod2 coding region pcr amplification result.

Fig. 5 cuts evaluation for the enzyme of recon pPICZ α A-sodM18.

Fig. 7 is the PCR qualification result of recombinant bacterial strain GS115/pPICZ α A-sodM18 genomic dna.

Fig. 9 is that the Native-PAGE of recombinant bacterial strain GS115/pPICZ α A-sodM18 expression product analyzes.

Figure 11 is that the SDS-PAGE of recombinant bacterial strain GS115/pPICZ α A-sodM18 expression product analyzes.

Figure 13 is a recombinant bacterial strain Mn-SOD enzyme activity determination.

Fig. 4 is CuZn-sod coding region pcr amplification result.

Fig. 6 cuts evaluation for the enzyme of recon pPICZ α A-sodC.

Fig. 8 is the PCR qualification result of recombinant bacterial strain GS115/pPICZ α A-sodC genomic dna.

Figure 10 is that the Native-PAGE of recombinant bacterial strain GS115/pPICZ α A-sodC expression product analyzes.

Figure 12 is that the SDS-PAGE of recombinant bacterial strain GS115/pPICZ α A-sodC expression product analyzes.

Figure 14 is a recombinant bacterial strain CuZn-SOD enzyme activity determination.

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment.

Embodiment 1, the secreting, expressing of Mn-sod2 gene in pichia spp

1, the structure that contains the carrier pPICZ α A-sodM18 of superoxide dismutase gene (Mn-sod2)

Structure contain superoxide dismutase gene carrier pPICZ α A-sodM18 process as shown in Figure 2, comprise the steps:

1) structure of carrier pET-22b-sod (building process is shown in Fig. 2 a): according to the suitable restriction enzyme site design primer of Mn-sod2 sequence and expression plasmid pPICZ α A, primer sequence is as follows:

Upstream primer P1:5 '--CG GAATTC(EcoRI) ATGTCTTCATTTCAATTGCC--3 '

Downstream primer P2:5 '--GTC GGTACC(KpnI) TGTTTTTGTGATTGAATTGC--3 '

Genomic dna with bacillus cereus M22 is a template, and under the guiding of primer P1 and primer P2, pcr amplification Mn-sod2 segment is used EcoRI and this gene fragment of XhoI double digestion again, at last it is used T ₄Dna ligase is connected with the carrier pET-22b that cuts with same enzyme enzyme, obtains containing the plasmid vector that is integrated with Mn-sod2, with its called after pET-22b-sod.

2) acquisition of goal gene Mn-sod2

The plasmid vector pET-22b-SOD that makes up with step 1) is a template, under the guiding of primer P1 and primer P2, carries out pcr amplification Mn-sod2, and makes clone's Mn-sod2 two ends add EcoR I and Kpn I recognition site respectively.The PCR product is detected through 1.2% agarose gel electrophoresis, and the result is (swimming lane 1 is a dna molecular amount standard, and swimming lane 2 is the PCR product) as shown in Figure 3, at about 630bp place a clear band is arranged, big or small consistent with Mn-sod2 conforms to expected results, showing that amplification has obtained correct Mn-sod2.

3) with restriction enzyme EcoR I and Kpn I double digestion carrier pPICZ α A (Invitrogen company).

4) the Mn-sod2 goal gene segment that obtains through pcr amplification with restriction enzyme EcoR I and Kpn I double digestion is then with itself and step 2) the carrier pPICZ α A T that cuts with same enzyme enzyme ₄Dna ligase connects, and obtains containing the recombinant vectors of Mn-sod2, called after pPICZ α A-sodM18.

2, the enzyme of recombinant plasmid pPICZ alpha A-sodM18 is cut evaluation

Recombinant vectors pPICZ α A-sodM18 with heat shock method transformed into escherichia coli DH5 α competent cell, was screened with 37 ℃ of cultivations of the less salt LB resistant panel that contains 25mg/mL Zeocin in 12-24 hour.Picking contains in the liquid LB substratum of 25ml/L Zeocin in 5ml at the single colony inoculation that grows on the above-mentioned resistant panel, cultivates 12-24 hour for 37 ℃, uses alkaline lysis method of extracting plasmid DNA, carries out restriction enzyme and cuts evaluation.With the EcoRI single endonuclease digestion and with EcoRI and KpnI double digestion pPICZ α A, pPICZ α A-sodM18, (

swimming lane

1,5 is Marker to the result as shown in Figure 5, swimming lane 2 is EcoRI, KpnI/pPICZ α A-sodM18, swimming lane 3 are EcoRI/pPICZ α A-sodM18, and swimming lane 4 is EcoRI/pPICZ α A), as can be seen,

swimming lane

3,4 all has only 1 band, and swimming lane 2 then has 2 bands, determines that the recombinant plasmid that is obtained is correct.

4, recombinant vectors pPICZ α A-sodM18 is transformed pichia spp

1) with plasmid linearization to be transformed

Because of the easier conversion of linearizing plasmid is advanced in the yeast chromosomal, earlier plasmid recombinant is carried out single endonuclease digestion, make it linearizing.Get 50 μ l plasmid pPICZA-sodM18 and cut 3h with the SacI enzyme.The enzyme system of cutting is:

Sterilized water is an amount of

DNA 50μl

10×buffer 40μl

SacI 100U

Cumulative volume 100 μ l

100 μ l enzymes are cut product use isopyknic phenol successively: chloroform, chloroform extracting, add the sodium acetate (pH5.2) of 1/10 volume and the dehydrated alcohol of 2-3 times of volume again and precipitate, wash once with 70% ethanol again, be dissolved in after the vacuum-drying in the 10 μ l water, stand-by.

2) preparation of pichia spp competent cell

Bacterial strain: pichia spp GS115

YPD (Yeast Extract Peptone Dextrose Mediun) solid medium (g/L): in the 850ml deionized water, add yeast extract 10, Tryptones 20, be settled to 900ml, add the 20g agar powder again, be cooled to and be lower than 100ml 20% glucose that adds 8 pounds of moist heat sterilizations after 50 ℃, the system of falling is dull and stereotyped, 4 ℃ of preservations.

YPD liquid nutrient medium (g/L): autoclaving, the cooling back adds 100ml 20% glucose of 8 pounds of moist heat sterilizations, 4 ℃ of preservations

(1) the single bacterium colony of the pichia spp GS115 on the picking YPD flat board is in the 5mlYPD liquid nutrient medium, and 30 ℃, 300rpm shaking table spend the night;

(2) the fresh GS115 bacterium liquid that shaking table is spent the night is forwarded among the 100mlYPD by 1/1000 inoculum size, and 30 ℃, 300rpm shaking table spend the night OD ₆₀₀When being about 1.3-1.5, the failure of oscillations;

(3) the bacterium liquid with precooling is transferred in the centrifuge tube of ice precooling, 4 ℃, the centrifugal 5min of 1500g, and precipitation is resuspended with the sterilized water of 100ml precooling;

(4) 4 ℃, the centrifugal 5min of 1500g, precipitation is resuspended with the sterilized water of 50ml precooling;

(5) 4 ℃, the centrifugal 5min of 1500g, precipitation is resuspended with the 1mol/L Sorbitol Solution USP of 4ml precooling;

(6) 4 ℃, the centrifugal 5min of 1500g precipitate resuspended, standby with the Sorbitol Solution USP of 200 μ l precoolings.

3) electric shock of plasmid transforms

Plasmid DNA: linearizing plasmid vector pPICZ α A-sodM18

Host bacterium: pichia spp GS115 competent cell

YPDS (Yeast Extract Peptone Dextrose Mediun With Sorbitol) solid medium (g/L):

In the 850ml deionized water, add yeast extract 10, Tryptones 20, sorbyl alcohol 182.2, agar 20 is settled to 900ml, autoclaving, the cooling back adds the 100ml20% glucose of 8 pounds of moist heat sterilizations, and the system of falling is dull and stereotyped, 4 ℃ of preservations.

(1) 80 μ l pichia spp GS115 competent cells is mixed with 10 μ l linearizings plasmid DNA to be transformed, and it is gone in the 0.2cm electricity conversion cup;

(2) the conversion cup ice bath 5min of mixed solution will be housed;

(3) parameter (electric capacity 20 μ F, voltage 2000V) of adjustment gene introducing apparatus shocks by electricity once, and the time is about 5ms;

(4) immediately toward transforming the 1mol/L Sorbitol Solution USP that adds the 1ml precooling in the cup, behind the mixing, it is gone in the centrifuge tube of sterilization;

(5) mixed solution is applied on the YPDS plate (containing 100mg/ml Zeocin), is coated with the mixed solution of 150 μ l-300 μ l on each YPDS plate;

(6) the YPDS plate is placed 30 ℃ of incubators cultivate 2-3d, till growing single bacterium colony.

5, the PCR of recombinant bacterial strain identifies

To transform pichia spp GS115 competent cell by electric shocking method through the linearizing pPICZ α of SacI single endonuclease digestion A-sodM18, (the Zeocin final concentration is respectively 100 to coat the YPDS flat board, 300,1000 μ g/mL), cultivate after 2-4 days for 28 ℃, picking list bacterium colony is cultivated, and extracts genomic dna, utilizes special primer P1 and P2 to carry out PCR and identifies.Result's swimming lane 1 as shown in Figure 7 is a dna molecular amount standard, swimming lane 2-5 is for being the PCR product of template with GS115/pPICZ α A-sod18 genomic dna, swimming lane 6 is for being that the PCR product of template is as negative control with GS115/pPICZ α A genomic dna), at about 630bp place a clear band is arranged, show that special primer P1 can conform to expected results from the dna fragmentation that positive recombinant bacterial strain amplification obtains about 630bp with P2, promptly conform to, illustrate that foreign gene successfully recombinated on the genome karyomit(e) of pichia spp with the size (627bp) of Mn-sod2 gene.

6, the abduction delivering of Mn-sod2 gene in pichia spp

YPDS (Yeast Extract Peptone Dextrose Mediun With Sorbitol) solid medium: in the 850ml deionized water, add yeast extract 10, Tryptones 20, sorbyl alcohol 182.2, agar 20, be settled to 900ml, autoclaving, the cooling back adds 100ml 20% glucose of 8 pounds of moist heat sterilizations, the system of falling is dull and stereotyped, 4 ℃ of preservations.

BMGY (Buferred Glycerol-complex Medium) selected liq substratum: in the 650ml deionized water, add yeast extract 10, Tryptones 20, be settled to 700ml after the dissolving, autoclaving, to be cooled to below 50 ℃ the time, adding is through 10 * YNB100ml of 0.22 μ m filtration sterilization, 500 * B 2ml, 10 * GY 100ml, 1M 100ml, 6.0,4 ℃ of preservations of pH.

500 * B (vitamin H): the 20mg vitamin H is dissolved in the 100ml deionized water, 0.22 μ m filtration sterilization behind the mixing, 4 ℃ of preservations, preservation period is about 1 year.

10 * YNB (yeast nitrogen base, sulfur-bearing acid amide do not contain amino acid): 134g yeast nitrogen base (the sulfur-bearing acid amide does not contain amino acid) is dissolved in the 1000ml deionized water, 0.22 μ m filtration sterilization behind the mixing, 4 ℃ of preservations, preservation period is about 1 year.

10 * M (methyl alcohol): 5ml methyl alcohol is joined in the 950ml deionized water, 0.22 μ m filtration sterilization behind the mixing, 4 ℃ of preservations, preservation period is about 2 months.

10 * GY (glycerine): 100ml glycerine is joined in the 1000ml deionized water, 0.22 μ m filtration sterilization or moist heat sterilization, room temperature preservation, preservation period are about 1 year.

1M phosphate buffered saline buffer (pH 6.0): in the 850ml deionized water, add 1M Na ₂HPO ₁12m, 1M NaH ₂PO ₄88ml.

(1) mono-clonal on picking YPDS (the containing 1000mg/mL Zecion) flat board is inoculated in the 50ml centrifuge tube that the 10mlBMGY substratum is housed, and 30 ℃, 250-280rpm shaking table were cultivated 2-3 days;

(2) shaking table is cultivated 2-3 days nutrient solution, and 4 ℃, the centrifugal 15min of 3000g gets precipitation (as far as possible supernatant being eliminated), and adding 40ml contains the BMMY substratum of 0.5% methyl alcohol in precipitate again, carries out inducing culture at 28 ℃, 250-280rpm;

(3) for the volatilization loss of compensation methyl alcohol, added methyl alcohol in per 24 hours, make the methanol concentration in the bacterium liquid remain on 0.5%.

(4) every the 24h sampling, 4 ℃, the centrifugal 5min of 12000g collects supernatant liquor, albumen to be detected and enzyme activity.

(5) induce 4-5 days after, stop to induce, 4 ℃, the centrifugal 5min of 12000g collects supernatant liquor, albumen to be detected and enzyme activity.

The detection of embodiment 2, Mn-sod2 gene secreting, expressing in pichia spp

Picking is accredited as male recombinant bacterial strain GS115/Mn-sod2 and changes empty plasmid control strain inoculation BMGY substratum, is cultured to OD ₆₀₀=2-6, behind the collection thalline, switching BMMY substratum carries out abduction delivering.

1, Native-PAGE electrophoretic analysis

Get respectively and induce yeast liquid preceding, that induce 24h, 48h, 72h, 4 ℃, the centrifugal 5min of 12000rpm get supernatant and carry out the Native-PAGE electrophoresis.(swimming lane 1 is for changeing empty plasmid control strain/72h as shown in Figure 9 for electrophoresis result, swimming lane 2 is before GS115/pPICZ α A-sodM18/ induces, swimming lane 3 is GS115/pPICZ α A-sodM18/24h, swimming lane 4 is GS115/pPICZ α A-sodM18/48h, swimming lane 5 is GS115/pPICZ α A-sodM18/72h), recombinant bacterial strain non-activity band on the Nativ-PAGE gel before the methanol induction occurs, and on the Native-PAGE gel behind the methanol induction, show single bright band, and brightness strengthens with the increase of induction time, and is the highest to 72 hours expression amounts.

2, SDS-PAGE electrophoretic analysis

With before inducing and induce the bacterium liquid supernatant behind the 72h to carry out SDS-PAGE, (swimming lane 1 is before GS115/pPICZ α A-sodM18/ induces to the result as shown in figure 11, swimming lane 2 is GS115/pPICZ α A-sodM18/72h), at about 23kD place one differential protein band is arranged, consistent with the theoretical molecular of inferring from aminoacid sequence.Illustrate that target Mn-sod2 gene realized secreting, expressing in pichia spp GS115.

3, the NBT method is measured the SOD enzyme assay

Learn from else's experience bacterium liquid behind the methanol induction is got supernatant after centrifugal, measures the activity of SOD enzyme in the supernatant liquor with NBT light inhibition method, draw 30 μ l samples, put into 10ml transparent glass small beaker, add 3ml NBT reaction solution, mixing, in 28 ℃ of SOD photochmeical reaction chambers, 2 * 15W fluorescent lamp illumination 20min measures absorbancy at the 560nm place, make blank with the NBT reaction solution, establish two repetitions for every group, whole mensuration process is all carried out under black out or low light condition except that photochmeical reaction.Be decided to be an enzyme activity unit with the 50% required enzyme amount that suppresses NBT photochemical reduction speed of reaction.Formula is as follows:

Sample enzyme activity (U/ml)=(OD _Contrast-OD _Sample)/(1/2OD _Contrast) * extension rate

The result shows that the activity of the SOD enzyme that the recombinant pichia yeast strain that is integrated with the Mn-sod2 gene is expressed is lower as shown in figure 13 before methanol induction, be about 12U/ml.Behind the methanol induction, the enzyme work of recombinant bacterial strain is about 43U/ml, than having improved about 2.6 times before inducing.

Embodiment 3, the secreting, expressing of CuZn-sod gene in pichia spp

1, the structure that contains the carrier pPICZ α A-sodC of superoxide dismutase gene (CuZn-sod)

Structure contain superoxide dismutase gene carrier pPICZ α A-sodC process as shown in Figure 2, concrete steps are as follows:

1) structure of carrier pET-22b-sod1: according to the suitable restriction enzyme site design primer of CuZn-sod sequence and expression plasmid pPICZ α A, primer sequence is as follows:

Upstream primer P3:5 '--CGC GAATTC(EcoRI) GGCAGAATATATTTTGGAGGG--3 '

Downstream primer P4:5 '--GTC GGTACC(KpnI) TTTTTCTTCATATCCGATGC--3 '

Genomic dna with bacillus cereus M22 is a template, and under the guiding of primer P3 and primer P4, pcr amplification CuZn-sod segment is used EcoRI and this gene fragment of Kpn I double digestion again, at last it is used T ₄Dna ligase is connected with the carrier pET-22b that cuts with same enzyme enzyme, obtains containing the plasmid vector that is integrated with CuZn-sod, with its called after pET-22b-sod1.

2) acquisition of goal gene CuZn-sod

Plasmid vector pET-22b-SOD1 with structure among the embodiment 1 is a template, under the guiding of primer P3 and primer P4, carries out pcr amplification CuZn-sod, and makes clone's CuZn-sod two ends add EcoR I and Kpn I recognition site respectively.The PCR product is detected through 1.2% agarose gel electrophoresis, (swimming lane 1 is a dna molecular amount standard to the result as shown in Figure 3, swimming lane 2 is the PCR product), at about 540bp place a clear band is arranged, big or small consistent with the CuZn-sod gene, conform to expected results, show that amplification has obtained correct CuZn-sod.

4) the CuZn-sod goal gene segment that obtains through pcr amplification with restriction enzyme EcoR I and Kpn I double digestion is then with itself and step 2) the carrier pPICZ α A T that cuts with same enzyme enzyme ₄Dna ligase connects, and obtains containing the recombinant vectors of CuZn-sod, called after pPICZ α A-sodC.

2, the enzyme of recombinant plasmid pPICZ alpha A-sodC is cut evaluation

Recombinant vectors pPICZ α A-sodC with thermal shock method transformed into escherichia coli DH5 α competent cell, was screened with 37 ℃ of cultivations of the less salt LB resistant panel that contains 25mg/mL Zeocin in 12-24 hour.Picking contains in the liquid LB substratum of 25ml/L Zeocin in 5ml at the single colony inoculation that grows on the above-mentioned resistant panel, cultivates 12-24 hour for 37 ℃, uses alkaline lysis method of extracting plasmid DNA, carries out restriction enzyme and cuts evaluation.With the EcoRI single endonuclease digestion and with EcoRI and KpnI double digestion pPICZ α A, pPICZ α A-sodC, (

swimming lane

1,5 is Marker to the result as shown in Figure 6, swimming lane 2 is EcoRI, KpnI/pPICZ α A-sodC, swimming lane 3 are EcoRI/pPICZ α A-sodC, and swimming lane 4 is EcoRI/pPICZ α A), as can be seen, swimming

lane

4, recombinant vectors pPICZ α A-sodC is transformed pichia spp

Method for transformation is with embodiment 1.

5, the PCR of recombinant bacterial strain identifies

To transform pichia spp GS115 competent cell by electric shocking method through the linearizing pPICZ α of SacI single endonuclease digestion A-sodC, (the Zeocin final concentration is respectively 100 to coat the YPDS flat board, 300,1000 μ g/mL), cultivate after 2-4 days for 28 ℃, picking list bacterium colony is cultivated, and extracts genomic dna, and be template with it, utilize universal primer P5, the P6 of special primer to P3, P4 and pichia spp

p5：5′--GACTGGTTCCAATTGACAAGC--3′

p6：5′--GCAAATGGCATTCTGACATCC--3′

Carrying out PCR identifies.(swimming lane M is a dna molecular amount standard to the result as shown in Figure 8, swimming lane 1 is with P3, P4 is the PCR product of primer, swimming lane 2 is with P5, P6 is the PCR product of primer), at about 630bp place a clear band is arranged, show that special primer P1 can conform to expected results from the dna fragmentation that positive recombinant bacterial strain amplification obtains about 630bp with P2, promptly conform to the size (540bp) of CuZn-sod gene, and universal primer P5 and P6 can be from increase dna fragmentations about 1140bp of recombinant bacterial strain, also conform to, show that foreign gene successfully recombinated on the genome karyomit(e) of pichia spp with expected results.

6, the abduction delivering of CuZn-sod gene in pichia spp

The method of abduction delivering is with embodiment 1.

The detection of embodiment 4, CuZn-sod gene secreting, expressing in pichia spp

Picking is accredited as male recombinant bacterial strain GS115/CuZn-sod2 and changes empty plasmid control strain inoculation BMGY substratum, is cultured to OD ₆₀₀=2-6, behind the collection thalline, switching BMMY substratum carries out abduction delivering.

1, Native-PAGE electrophoretic analysis

Get respectively and induce preceding, as to induce 72h yeast liquid, 4 ℃, the centrifugal 5min of 12000rpm get supernatant and carry out the Native-PAGE electrophoresis.Electrophoresis result is (preceding swimming lane 2 is GS115/pPICZ α A-sodC/72h to swimming lane 1 for GS115/pPICZ α A-sodC/ induces) as shown in figure 10, recombinant bacterial strain non-activity band on the Nativ-PAGE gel before the methanol induction occurs, and on the Native-PAGE gel behind the methanol induction, show single bright band, and on the Native-PAGE gel behind the methanol induction, demonstrate single bright band, illustrate through abduction delivering to have gone out activated albumen.

2, SDS-PAGE electrophoretic analysis

With before inducing and induce the bacterium liquid supernatant behind the 72h to carry out SDS-PAGE, (swimming lane 1 is before GS115/pPICZ α A-sodC/ induces to the result as shown in figure 12, swimming lane 2 is GS115/pPICZ α A-sodC/72h), at about 23kD place one differential protein band is arranged, consistent with the theoretical molecular of inferring from aminoacid sequence.Illustrate that target Mn-sod2 gene realized secreting, expressing in pichia spp GS115.

3, the NBT method is measured the SOD enzyme assay

The result shows that the activity of the SOD enzyme that the recombinant pichia yeast strain that is integrated with the CuZn-sod gene is expressed is lower as shown in figure 14 before methanol induction, be about 15U/ml.Behind the methanol induction, the enzyme work of recombinant bacterial strain is about 35U/ml, than having improved about 1.3 times before inducing.

Claims

1, a kind of carrier of expressing superoxide dismutase gene contains the yeast expression vector that superoxide dismutase gene, AOX1 promotor and terminator sequence, multiple clone site and screening indicate.

2, carrier according to claim 1 is characterized in that: described superoxide dismutase gene is between the EcoR I and Kpn I restriction enzyme digestion sites at the multiple clone site place of yeast expression vector.

3, dedicated carrier according to claim 1 and 2 is characterized in that: described screening is masked as Zeocin ^RThe screening sign.

4, according to claim 1 or 2 or 3 described carriers, it is characterized in that: described superoxide dismutase gene is Mn-SOD or CuZn-SOD gene.

5, dedicated carrier according to claim 4 is characterized in that: the described yeast expression vector that contains superoxide-dismutase base Mn-SOD gene is pPICZ α A-sodM18; The described yeast expression vector that contains superoxide-dismutase base CuZn-SOD gene is pPICZ α A-sodC.

6, a kind of method of superoxide dismutase gene expression, be to make up the yeast expression vector that contains superoxide dismutase gene, the yeast expression vector that makes up is imported in the pichia spp, obtain recombinant yeast pichia pastoris, induce recombinant yeast pichia pastoris to make superoxide dismutase gene obtain expressing.

7, the method for superoxide dismutase gene expression according to claim 6 is characterized in that: the described method that the yeast expression vector that makes up is imported in the pichia spp is an electrotransformation.

8, the method expressed of superoxide dismutase gene according to claim 7 is characterized in that: describedly add methyl alcohol when inducing restructured Pichia pastoris in expression, the concentration of methyl alcohol remains on 0.4-0.6%.

9, the method expressed of superoxide dismutase gene according to claim 7 is characterized in that: described to induce the time of restructured Pichia pastoris in expression be 2-6 days.