CN111378676B - Construction and application of pCUP1 vector plasmid - Google Patents

Abstract

The invention provides a construction method of pCUP1 carrier plasmid and a screening method of recombinant pichia pastoris with exogenous gene high copy and stable insertion; wherein the construction method of the pCUP1 vector plasmid comprises the following steps: amplifying a CUP1 gene in the saccharomyces cerevisiae by adopting a PCR amplification system to obtain a PCR product, and recovering the PCR product by adopting electrophoresis to obtain a CUP1 gene fragment; carrying out enzyme digestion on the pPICZ alpha A vector by using BamH I restriction endonuclease to obtain an enzyme digestion product, and recovering the enzyme digestion product by using electrophoresis to obtain the enzyme digested pPICZ alpha A vector; and (3) connecting the CUP1 gene fragment with the digested pPICZ alpha A vector to obtain the pCUP1 vector plasmid. The recombinant pichia pastoris screening method for exogenous gene high-copy stable insertion of the pCUP1 carrier plasmid is adopted, and pichia pastoris is promoted to spontaneously form high-copy exogenous gene insertion under the induction action of copper ions with different concentrations, so that the recombinant pichia pastoris screening method for exogenous gene high-copy stable insertion with short screening period, high efficiency and low cost is achieved.

Description

Construction and application of pCUP1 vector plasmid

Technical Field

The invention belongs to the technical field of biology, and particularly relates to construction of a pCUP1 vector plasmid, a method for screening recombinant pichia pastoris with high copy number and foreign gene insertion by adopting the pCUP1 vector plasmid, and application of the method.

Background

The pichia pastoris recombinant expression system is a widely applied recombinant protein expression system. Among them, the most commonly used inducible expression scheme using a methanol promoter is the following process: the exogenous gene is inserted into expression plasmids such as pPIC9k, pPICZ alpha A and pPIC3.5k with AOX1 promoters, and then the linearized expression plasmids are transformed into pichia pastoris competent cells by an electrotransformation method.

In the methods, because the used expression plasmids have sequences which are homologous with host genes, the exogenous plasmids can be recombined with the homologous genes in the host, so that the exogenous genes can be stably integrated into a pichia pastoris genome, and Zeocin or g418 resistance genes carried on the expression plasmids are simultaneously integrated into the pichia pastoris genome to realize constitutive expression, and then a resistance screening method can be used for identifying whether the clone with the exogenous genes is correctly inserted. In these screening methods, the concentration of the antibiotic resistant transformant is positively correlated with the integrated copy number of the foreign gene in the transformant within a certain range, so that clones into which foreign genes of different copy numbers are inserted can be screened by using antibiotic plates of different concentrations.

Further, homologous integration of the pPIC9k, ppicza, ppic3.5k plasmids with high copy integration event is a random low probability event. Generally, in order to select transformants with high copy inserted foreign gene, a large number of transformants are selected one by using Zeocin or g418 antibiotic plates with low to high concentration, and finally, the clone which can survive in the high concentration antibiotic plate is the transformant with multi-copy inserted foreign gene. The method has the defects that hundreds or even thousands of transformants are usually screened by the method, and only a few high-copy clones can be obtained by screening 4-5 plates with different concentrations in sequence, so that the efficiency is low; in addition, pichia pastoris has strong tolerance to g418 antibiotics, false positive is easy to occur in the screening process, and the correlation between the expression quantity and the drug concentration is not obvious. Moreover, Zeocin reagent is expensive, it is not easy to screen for high expressing strains at low concentrations, and it is extremely easy to kill all cloned products at high concentrations.

Disclosure of Invention

In view of the above problems, the present invention aims to establish a pCUP1 vector plasmid with a copper-resistant gene (CUP1 gene), and to utilize the CUP1 gene to promote pichia pastoris to spontaneously form high-copy foreign gene insertion under the induction action of copper ions of different concentrations, so as to achieve a recombinant pichia pastoris screening method with short screening period, high efficiency and low cost for stably inserting the foreign gene with high copy.

The technical scheme for realizing the purpose is as follows:

the invention provides a construction method of pCUP1 carrier plasmid, which comprises the following steps:

amplifying a CUP1 gene in the saccharomyces cerevisiae by adopting a PCR amplification system to obtain a PCR product, and recovering the PCR product by adopting electrophoresis to obtain a CUP1 gene fragment;

carrying out enzyme digestion on the pPICZ alpha A vector by using BamH I restriction endonuclease to obtain an enzyme digestion product, and recovering the enzyme digestion product by using electrophoresis to obtain the enzyme digested pPICZ alpha A vector;

connecting the CUP1 gene segment with the digested pPICZ alpha A vector to obtain the pCUP1 vector plasmid (the sequence table is shown in the attached part);

wherein the content of the first and second substances,

the amplification primers in the PCR amplification system are as follows:

an upstream primer F: at the time of the start of the 5' TGAGACCTTCGTTTGTGCG caatagaggcaggtatc,

a downstream primer R: 5' ATGGTGTGTGGGGGATC attcctttaattgctaa;

the PCR amplification system is 100ul, and comprises:

10 Xamplification buffer solution 10ul, dATP 200umol/L, dGTP 200umol/L, dTTP 200umol/L, dCTP 200umol/L, the upstream primer F10-100 pmol, the downstream primer R10-100 pmol, template DNA 0.1-2 ug, Taq DNA polymerase 2.5u, Mg²⁺1.5mmol/L, and the balance of double distilled water and/or triple distilled water;

the reaction conditions of the PCR amplification system are as follows:

sequentially denaturing at 94 ℃ for 60 seconds, annealing at 55-60 ℃ for 60 seconds, derivatizing at 72 ℃ for 120 seconds, and repeating for 25-30 cycles.

In one embodiment, in the construction method of pCUP1 vector plasmid, the electrophoretic recovery is agarose nucleic acid electrophoretic recovery;

preferably, the pPICZ alpha A vector is subjected to enzyme digestion by using BamH I restriction endonuclease to obtain an enzyme digestion product; recovering the enzyme digestion product by adopting electrophoresis to obtain the pPICZ alpha A vector after enzyme digestion, comprising the following steps: mixing 2-5 mu g of pPICZ alpha A vector with 20-50u of BamH I restriction endonuclease to obtain a mixture; performing enzyme digestion on the mixture at 37 ℃ overnight to obtain an enzyme digestion product, and then recovering the enzyme digestion product by agarose nucleic acid electrophoresis to obtain the pPICZ alpha A vector after enzyme digestion;

preferably, the step of linking the CUP1 gene fragment with the cleaved pPICZ alpha A vector to obtain the pCUP1 vector plasmid comprises: and adopting an easy Geno rapid recombinant cloning kit to connect the CUP1 gene fragment with the digested pPICZ alpha A vector to obtain the pCUP1 vector plasmid.

The invention also provides a screening method of recombinant pichia pastoris with high copy and stable insertion of exogenous genes, which adopts the pCUP1 vector plasmid, and the screening method comprises the following steps:

carrying out double enzyme digestion on the pCUP1 carrier plasmid by using EcoR I restriction endonuclease and Not I restriction endonuclease to obtain an enzyme digestion product, and then recovering the enzyme digestion product by using electrophoresis to obtain the pCUP1 carrier plasmid after enzyme digestion;

adopting a PCR amplification system to amplify the exogenous gene to obtain an exogenous gene PCR product, and then adopting electrophoresis to recover the exogenous gene PCR product to obtain an exogenous gene fragment;

connecting the exogenous gene fragment with the pCUP1 vector plasmid after enzyme digestion to obtain the pCUP 1-exogenous gene expression plasmid;

linearizing the pCUP 1-exogenous gene expression plasmid by adopting Sac I restriction endonuclease to obtain a linearized pCUP 1-exogenous gene expression plasmid;

transforming X33 pichia pastoris competent cells by adopting the linearized pCUP 1-exogenous gene expression plasmid, then coating a YPDS-Zeocin plate, and culturing to obtain a first colony;

inoculating the first colony to a first BMGY liquid culture medium to obtain a culture, and inoculating the culture to a second BMGY liquid culture medium to obtain a bacterial liquid;

re-suspending the bacteria solution by using a third BMGY culture medium, coating a third BMGY flat plate, and culturing to obtain a recombinant pichia pastoris high-copy bacterial colony;

wherein the content of the first and second substances,

the YPDS-Zeocin flat plate comprises: yeast extract with concentration of 1%, peptone with concentration of 2%, glucose with concentration of 2%, sorbitol with concentration of 1mol/L, agar with concentration of 2%, Zeocin with concentration of 100 ug/ml;

the first BMGY liquid culture medium comprises: 1% yeast extract, 2% peptone, 100mM potassium dihydrogen phosphate, pH 6.0, 1.34% YNB, 4X10^-5% biotin, glycerol at a concentration of 1%, copper sulfate at a concentration of 0.3 mM;

the second BMGY liquid culture medium comprises: 1% yeast extract, 2% peptone, 100mM potassium dihydrogen phosphate, pH 6.0, 1.34% YNB, 4X10^-5% biotin, glycerol at a concentration of 1%, copper sulfate at a concentration of 1 mM;

the third BMGY culture medium comprises: 1% yeast extract, 2% peptone, 100mM potassium dihydrogen phosphate, pH 6.0, 1.34% YNB, 4X10^-5% biotin, glycerol at a concentration of 1%, copper sulfate at a concentration of 2 mM;

the third BMGY flat plate comprises: 1% yeast extract, 2% peptone, 1.5% agarose, 100mM potassium dihydrogen phosphate, pH 6.0, 1.34% YNB, 4X10^-5% biotin, glycerol at a concentration of 1% and copper sulphate at a concentration of 2 mM.

In one embodiment, the method for screening recombinant pichia pastoris with high copy number and stable insertion of foreign genes, provided by the invention, comprises the following steps of performing double enzyme digestion on the pCUP1 vector plasmid by using EcoR I restriction endonuclease and Not I restriction endonuclease to obtain an enzyme digestion product, and recovering the enzyme digestion product by using electrophoresis to obtain the enzyme digestion pCup1 vector plasmid, wherein the method comprises the following steps: mixing 2-5 mu g of pCUP1 carrier plasmid, 5u of EcoR I restriction endonuclease and 20-50u of Not I restriction endonuclease to obtain a mixture; carrying out enzyme digestion on the mixture at 37 ℃ overnight to obtain an enzyme digestion product, and then recovering the enzyme digestion product by adopting agarose nucleic acid electrophoresis to obtain a pCUP1 carrier plasmid after enzyme digestion;

preferably, the amplifying the exogenous gene by using the PCR amplification system to obtain an exogenous gene PCR product, and then recovering the exogenous gene PCR product by using electrophoresis to obtain an exogenous gene fragment, includes: amplifying the EGFP gene by adopting a PCR amplification system to obtain an EGFP gene PCR product, and then recovering the EGFP gene PCR product by adopting agarose nucleic acid electrophoresis to obtain an EGFP fragment (the sequence table is shown as an accessory);

preferably, the step of connecting the exogenous gene fragment with the pCUP1 vector plasmid after enzyme digestion to obtain the pCUP 1-exogenous gene expression plasmid comprises the following steps: connecting the EGFP fragment with the pCUP1 vector plasmid after enzyme digestion to obtain the pCUP1-EGFP expression plasmid (the sequence table is shown as an attachment);

preferably, the linearization of the pCUP 1-foreign gene expression plasmid using Sac I restriction endonuclease to obtain linearized pCUP 1-foreign gene expression plasmid comprises: linearizing the pCUP1-EGFP expression plasmid by adopting Sac I restriction endonuclease to obtain a linearized pCUP1-EGFP expression plasmid;

preferably, the linearized pCUP 1-exogenous gene expression plasmid is used for transforming X33 Pichia pastoris competent cells, YPDS-Zeocin plates are coated, and the colonies are obtained by culture, and comprise: and transforming X33 pichia pastoris competent cells by adopting the linearized pCUP1-EGFP expression plasmid, then coating a YPDS-Zeocin plate, and culturing to obtain a colony.

In one embodiment, in the method for screening recombinant pichia pastoris with high copy number and stable insertion of foreign genes, amplification primers in a PCR amplification system are as follows:

EGFP-F：5'agagaggctgaagct atggtgagcaag，

EGFP-R：5'agaaagctggcggccttacttgtacagct；

preferably, the PCR amplification system is 100ul, which comprises:

10 Xamplification buffer solution 10ul, dATP 200umol/L, dGTP 200umol/L, dTTP 200umol/L, dCTP 200umol/L, the upstream primer F10-100 pmol, the downstream primer R10-100 pmol, template DNA 0.1-2 ug, Taq DNA polymerase 2.5u, Mg²⁺1.5mmol/L, and the balance of double distilled water and/or triple distilled water;

the reaction conditions of the PCR amplification system are as follows:

sequentially denaturing at 94 ℃ for 60 seconds, annealing at 55-60 ℃ for 60 seconds, derivatizing at 72 ℃ for 120 seconds, and repeating for 25-30 cycles.

In one embodiment, in the method for screening recombinant pichia pastoris with high copy number and stable insertion of foreign genes, the step of connecting the EGFP fragment with the digested pCUP1 vector plasmid to obtain the pCUP1-EGFP fragment expression plasmid includes: connecting the EGFP fragment with the pCUP1 carrier plasmid after enzyme digestion by adopting an easy Geno rapid recombinant cloning kit to obtain the pCUP1-EGFP expression plasmid;

preferably, the linearization of the pCUP1-EGFP expression plasmid using Sac I restriction endonuclease to obtain a linearized pCUP1-EGFP expression plasmid comprises the following steps:

(1) mixing the Sac I restriction endonuclease with the pCUP1-EGFP expression plasmid; preferably, said Sac I restriction endonuclease 100u is mixed with 10. mu.g of said pCUP1-EGFP expression plasmid;

(2) digesting the mixture obtained in the step (1) at 37 ℃ overnight;

(3) reacting the reactant obtained in the step (2) at the temperature of 95 ℃ for 10 minutes, and then stopping the reaction;

(4) adding a sodium acetate solution with the concentration of 3M and absolute ethyl alcohol into the reactant obtained in the step (3), centrifuging, and taking a precipitate;

preferably, the volume ratio of the reactant obtained in the step (3) to the sodium acetate solution is 10 (0.5-1.5);

preferably, the volume ratio of the reactant obtained in the step (3) to the absolute ethyl alcohol is 1 (2-3);

(4) washing the precipitate obtained in the step (3) by using an ethanol solution with the mass concentration of 70%, centrifuging, taking the precipitate, and drying to obtain the linearized pCUP1-EGFP expression plasmid.

In one embodiment, in the method for screening recombinant pichia pastoris with high copy number and stable insertion of foreign genes, the linearized pCUP1-EGFP expression plasmid is adopted to transform X33 pichia pastoris competent cells, and then a YPDS-Zeocin plate is coated and cultured to obtain a first colony, which comprises the following steps:

(1) mixing 10 μ g of the linearized pCUP1-EGFP expression plasmid with 100 μ L of the Pichia X33 competent cells;

(2) keeping the mixture obtained in the step (1) for 5-30 min under an ice bath condition, and then carrying out electric shock; preferably, the condition of the electric shock is: the voltage is 1.5kV, the capacitance is 25 muF, the resistance is 200-400 omega, and the electric shock time is 10 msec;

(3) adding the reactant obtained in the step (2) into a sorbitol solution with the concentration of 0.5-1.5M and the temperature of 4 ℃;

(4) spreading the mixture obtained in step (3) on the YPDS-Zeocin plate, and culturing at 30 ℃ until the first colony appears.

In one embodiment, the method for screening recombinant pichia pastoris with exogenous genes inserted stably in high copy number comprises the following steps of:

(1) inoculating the first colony to the first BMGY liquid culture medium, and then performing shake culture for 11-13 h at the temperature of 30 ℃ and the speed of 200 rpm;

(2) and (2) inoculating the culture obtained in the step (1) to the second BMGY liquid culture medium, performing shaking culture for 23-25 h, centrifuging, and taking supernatant to obtain the bacterial liquid.

In one embodiment, in the method for screening recombinant pichia pastoris with high copy and stable insertion of foreign genes, the method comprises the following steps of resuspending the bacterial liquid by using a third BMGY culture medium, coating a BMGY plate, and culturing to obtain a recombinant pichia pastoris high copy colony, wherein the method comprises the following steps: and (3) resuspending the bacterial liquid by using a third BMGY culture medium, coating a BMGY flat plate, and performing static culture at the temperature of 30 ℃ until monoclone appears, thus obtaining the recombinant pichia pastoris with high copy number and exogenous gene insertion.

The invention also provides application of the pCUP1 vector plasmid and the recombinant pichia pastoris with high copy number and stable insertion of the exogenous gene in constructing the recombinant pichia pastoris with high copy number and inserted the exogenous gene, expressing the recombinant protein and purifying the recombinant protein.

Positive clones were identified by colony PCR method and gene sequencing to obtain the pCUP1 vector plasmid with completely correct circularization of the sequence, as shown in FIG. 1.

Specifically, in the invention, copper ions are used for inducing copy number mutation of pichia pastoris so as to screen recombinant pichia pastoris with high copy number inserted with exogenous genes, and the method specifically comprises the following steps:

(1) scraping a first colony (lawn) on the YPDS-Zeocin plate, inoculating the first colony (lawn) to the first BMGY liquid culture medium (containing copper sulfate with the concentration of 0.3 mM), and performing shake culture at the temperature of 30 ℃ and the speed of 200rpm for 11-13 h; inoculating the obtained culture to the second BMGY liquid culture medium (containing copper sulfate with the concentration of 1 mM), continuously culturing for 23-25 h, centrifuging, and taking the supernatant to obtain the bacterial liquid;

(2) collecting the obtained bacterial liquid, re-suspending the bacterial liquid by using the third BMGY culture medium (containing copper sulfate with the concentration of 2 mM), coating a BMGY plate (containing copper sulfate with the concentration of 2 mM), and culturing until monoclone appears, wherein the grown monoclone is a colony which is resistant to copper ions (containing a clone with the copy number increased by CUP 1), namely obtaining the recombinant pichia pastoris with high copy number inserted with the exogenous gene;

(3) and (3) selecting the third BMGY culture medium with the monoclonal appeared, carrying out shake culture until OD600 reaches 1-2, centrifuging, replacing with a BMMY culture medium, inducing for 48-72 hours, supplementing methanol every 24 hours, keeping the final concentration of methanol at 0.5%, and finishing the induced expression of the foreign protein.

Wherein the BMMY medium comprises: 1% yeast extract, 2% peptone, 100mM potassium dihydrogen phosphate, pH 6.0, 1.34% YNB, 4X10^-5% biotin, methanol at a concentration of 0.5%, copper sulfate at a concentration of 2 mM;

(4) detecting the contents of the colony screened by a conventional method and the EGFP gene of the recombinant pichia pastoris with high copy number and inserted with exogenous genes, which is obtained by the invention, by a fluorescent quantitative RT-PCR method; the detection result shows that under the induction action of copper ions, high-copy foreign gene insertion spontaneously formed by pichia pastoris can occur, the copy number of foreign gene (EGFP) expression near a CUP1 locus is amplified together, and the high-copy-number clone can be screened out under the high copper ion concentration.

Specifically, in the above fluorescent quantitative RT _ PCR method, the primers are:

EGFP-L：5'AAGGGCATCGACTTCAAGGA，

EGFP-R：5'GGTGTTCTGCTGGTAGTGGT；

selecting 30 single colonies obtained by the prior art after being screened by pPICZ alpha-EGFP/x 33 Zeocin, selecting 30 recombinant Pichia pastoris high-copy single colonies obtained by the screening method, extracting yeast genome DNA by adopting a commercial yeast genome DNA extraction kit, determining OD280 and OD260, and taking 20 samples with DNA quality close to each other to dilute the samples to the same concentration;

in the fluorescent quantitative RT _ PCR method, the reaction system is 25ul, which comprises: 2 x amplification buffer 12.5ul, primer EGFP-L (10 uM concentration) 10ul, primer EGFP-R (10 uM concentration) 10ul, polymerase 0.5u, template DNA100pg-100ng, and the balance deionized water.

In the fluorescent quantitative RT _ PCR method, the reaction conditions are as follows: sequentially modifying at 95 ℃ for 5min, at 95 ℃ for 10 s, annealing at 60 ℃ for 45 s, and repeating for 30-40 cycles; wherein the dissolution curve analysis conditions are as follows: in this order at a temperature of 95 ℃ for 15 seconds, at a temperature of 60 ℃ for 1min, at a temperature of 95 ℃ for 15 seconds, and at a temperature of 60 ℃ for 15 seconds.

According to the invention, by constructing a pCUP1 carrier plasmid with a CUP1 gene and transferring an exogenous gene (such as EGFP) into an X33 Pichia pastoris expression system through the pCUP1 carrier plasmid, the gene can spontaneously mutate in copy number during screening by utilizing the action that the CUP1 gene can be replicated, and the more the copy number is, the higher the expression amount of a target protein is. The screening method can obtain the recombinant pichia pastoris with high copy number inserted with the exogenous gene through the application of the pCUP1-EGFP expression plasmid and a pressure screening mode, thereby realizing the high expression of the exogenous gene in the pichia pastoris.

The protein encoded by the CUP1 in the pichia pastoris belongs to metallothionein which is rich in cysteine, and the CUP1 protein can chelate metal copper ions, so that the pichia pastoris has tolerance to the copper ions. In addition, the CUP1 gene is an essential gene for the survival of pichia pastoris in a high-copper environment, and the pichia pastoris generally has 2-15 copy numbers of CUP1 genes. Further, if the concentration of copper ions in the pichia pastoris living environment is increased, the copy number of the CUP1 gene will be increased, and finally, the cells which obtain more copy number of the CUP1 gene grow better.

Through a large number of screening tests, the invention discovers that after a plasmid with a copper-resistant gene (CUP1 gene) is transformed into wild Pichia pastoris X33, the plasmid can be integrated into a yeast chromosome, copper ions are added into a culture medium to promote copy number mutation of a CUP1 gene to form a CUP1 multi-copy mutant, the copy numbers of foreign gene expression elements near a CUP1 gene locus are amplified together, and a strain surviving from the high copper ions is a high-copy foreign gene strain.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows the pCUP1 vector plasmid constructed in the present invention;

FIG. 2 shows the pCUP1-EGFP expression plasmid constructed according to the present invention.

FIG. 3 shows the results of detection and analysis of recombinant Pichia pastoris with high copy number inserted with foreign genes, obtained by a conventional screening method and the screening method of the present invention in the examples of the present invention.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagent materials used in the following examples are all commercially available products unless otherwise specified.

Saccharomyces cerevisiae, pPICZ. alpha.A vector, BamH I restriction endonuclease, EasyGeno rapid recombinant cloning kit, EcoR I restriction endonuclease, Not I restriction endonuclease and X33 Pichia competent cells (prepared according to conventional techniques) used in the following examples were all commercially available products.

In the following examples;

the YPDS-Zeocin flat plate comprises: yeast extract with concentration of 1%, peptone with concentration of 2%, glucose with concentration of 2%, sorbitol with concentration of 1mol/L, agar with concentration of 2%, Zeocin with concentration of 100 ug/ml;

the first BMGY liquid culture medium comprises: 1% yeast extract, 2% peptone, 100mM potassium dihydrogen phosphate, pH 6.0, 1.34% YNB, 4X10^-5% biotin, glycerol at a concentration of 1%, copper sulfate at a concentration of 0.3 mM;

the second BMGY liquid culture medium comprises: 1% yeast extract, 2% peptone, 100mM potassium dihydrogen phosphate, pH 6.0, 1.34% YNB, 4X10^-5% biotin, glycerol at a concentration of 1%, copper sulfate at a concentration of 1 mM;

the third BMGY culture medium comprises: 1% concentration of yeast extract, concentratedPeptone 2% in concentration, potassium dihydrogen phosphate at 100mM, pH 6.0, YNB 1.34% in concentration, 4X10 in concentration^-5% biotin, glycerol at a concentration of 1%, copper sulfate at a concentration of 2 mM;

the third BMGY flat plate comprises: 1% yeast extract, 2% peptone, 100mM potassium dihydrogen phosphate, pH 6.0, 1.34% YNB, 4X10^-5% biotin, glycerol at a concentration of 1% and copper sulphate at a concentration of 2 mM.

Example 1: the invention relates to a method for screening recombinant pichia pastoris with exogenous genes inserted stably in high copy number

Firstly, constructing the pCUP1 carrier plasmid

(i) Amplifying a CUP1 gene in the saccharomyces cerevisiae by adopting a PCR amplification system to obtain a PCR product, and recovering the PCR product by adopting agarose nucleic acid electrophoresis to obtain a CUP1 gene fragment;

the amplification primers in the PCR amplification system are as follows:

an upstream primer F: at the time of the start of the 5' TGAGACCTTCGTTTGTGCG caatagaggcaggtatc,

a downstream primer R: 5' ATGGTGTGTGGGGGATC attcctttaattgctaa;

the PCR amplification system is 100ul, and comprises:

10 Xamplification buffer 10ul, dATP 200umol/L, dGTP 200umol/L, dTTP 200umol/L, dCTP 200umol/L, the upstream primer F10pmol, the downstream primer R10pmol, template DNA0.1ug, Taq DNA polymerase 2.5u, Mg²⁺1.5mmol/L, and the balance of double distilled water and/or triple distilled water;

the reaction conditions of the PCR amplification system are as follows:

denaturation at 94 ℃ for 60 seconds, annealing at 55-60 ℃ for 60 seconds, derivatization at 72 ℃ for 120 seconds, and repeating for 25 cycles in this order.

(ii) Mixing 2 mu g of pPICZ alpha A vector with 20u of BamH I restriction endonuclease to obtain a mixture; and carrying out enzyme digestion on the mixture at 37 ℃ overnight to obtain an enzyme digestion product, and then recovering the enzyme digestion product by adopting agarose nucleic acid electrophoresis to obtain the pPICZ alpha A vector after enzyme digestion.

(iii) And (3) connecting the CUP1 gene fragment with the cut pPICZ alpha A vector by adopting an easy Geno rapid recombinant cloning kit to obtain the pCUP1 vector plasmid (the sequence table is shown in an attachment).

Secondly, constructing the pCUP1-EGFP expression plasmid

(i) Mixing 2 mu g of pCUP1 carrier plasmid, EcoR I restriction endonuclease 5u and Not I restriction endonuclease 20u to obtain a mixture; and carrying out enzyme digestion on the mixture at 37 ℃ overnight to obtain an enzyme digestion product, and then recovering the enzyme digestion product by adopting agarose nucleic acid electrophoresis to obtain the pCUP1 vector plasmid after enzyme digestion.

(ii) Amplifying the EGFP gene by adopting a PCR amplification system to obtain an EGFP gene PCR product, and then recovering the EGFP gene PCR product by adopting agarose nucleic acid electrophoresis to obtain an EGFP fragment (the sequence table is shown as an attachment).

(iii) And (3) connecting the EGFP fragment with the pCUP1 vector plasmid after enzyme digestion by adopting an easy Geno rapid recombinant cloning kit to obtain the pCUP1-EGFP expression plasmid (the sequence table is shown as an attachment).

Thirdly, screening recombinant pichia pastoris with high copy number and foreign gene insertion

(i) Linearizing the pCUP1-EGFP expression plasmid by using Sac I restriction endonuclease to obtain a linearized pCUP1-EGFP expression plasmid, comprising the following steps: (1) mixing said Sac I restriction endonuclease 100u with 10 ug of said pCUP1-EGFP expression plasmid; (2) digesting the mixture obtained in the step (1) at 37 ℃ overnight; (3) reacting the reactant obtained in the step (2) at the temperature of 95 ℃ for 10 minutes, and then stopping the reaction; (4) adding a sodium acetate solution with the concentration of 3M and absolute ethyl alcohol into the reactant obtained in the step (3), centrifuging, and taking a precipitate; the volume ratio of the reactant obtained in the step (3) to the sodium acetate solution is 10: 0.5; the volume ratio of the reactant obtained in the step (3) to the absolute ethyl alcohol is 1: 2; (4) washing the precipitate obtained in the step (3) by using an ethanol solution with the mass concentration of 70%, centrifuging, taking the precipitate, and drying to obtain the linearized pCUP1-EGFP expression plasmid.

(ii) Transforming X33 pichia pastoris competent cells by adopting the linearized pCUP1-EGFP expression plasmid, then coating a YPDS-Zeocin plate, and culturing to obtain a first colony, wherein the method comprises the following steps: (1) mixing 10 μ g of the linearized pCUP1-EGFP expression plasmid with 100 μ L of the Pichia X33 competent cells; (2) keeping the mixture obtained in the step (1) for 5min under an ice bath condition, and then carrying out electric shock; the conditions of the electric shock are as follows: the voltage was 1.5kV, the capacitance was 25. mu.F, the resistance was 200. omega. and the shock time was 10 msec. (3) Adding the reactant obtained in the step (2) into a sorbitol solution with the concentration of 0.5M and the temperature of 4 ℃; (4) spreading the mixture obtained in step (3) on the YPDS-Zeocin plate, and culturing at 30 ℃ until the first colony appears.

(iii) Inoculating the first colony on a first BMGY liquid medium (containing copper sulfate with the concentration of 0.3 mM) to obtain a culture, and inoculating the culture on a second BMGY liquid medium (containing copper sulfate with the concentration of 1 mM) to obtain a bacterial liquid, wherein the method comprises the following steps of: (1) inoculating the first colony to the first BMGY liquid culture medium, and then performing shake culture for 11h at the temperature of 30 ℃ and the speed of 200 rpm; (2) and (2) inoculating the culture obtained in the step (1) to the second BMGY liquid culture medium, performing shaking culture for 23h, centrifuging, and taking the supernatant to obtain the bacterial liquid.

(iv) And (3) resuspending the bacterial liquid by using the third BMGY culture medium (containing copper sulfate with the concentration of 2 mM), coating the BMGY plate (containing copper sulfate with the concentration of 2 mM), and standing and culturing at the temperature of 30 ℃ until monoclonals appear, wherein the grown clones are colonies resistant to copper ions (containing clones with increased copy number of CUP 1), and thus obtaining the recombinant pichia pastoris with high copy number inserted with the foreign gene.

(v) And (3) selecting the third BMGY culture medium with the monoclonal appeared, carrying out shake culture until OD600 reaches 1-2, centrifuging, replacing with a BMMY culture medium, inducing for 48-72 hours, supplementing methanol every 24 hours, keeping the final concentration of methanol at 0.5%, and finishing the induced expression of the foreign protein.

Practice ofExample 2: the invention relates to a method for screening recombinant pichia pastoris with exogenous genes inserted stably in high copy number

Firstly, constructing the pCUP1 carrier plasmid

(i) Amplifying a CUP1 gene in the saccharomyces cerevisiae by adopting a PCR amplification system to obtain a PCR product, and recovering the PCR product by adopting agarose nucleic acid electrophoresis to obtain a CUP1 gene fragment;

the amplification primers in the PCR amplification system are as follows:

an upstream primer F: at the time of the start of the 5' TGAGACCTTCGTTTGTGCG caatagaggcaggtatc,

a downstream primer R: 5' ATGGTGTGTGGGGGATC attcctttaattgctaa;

the PCR amplification system is 100ul, and comprises:

10 Xamplification buffer 10ul, dATP 200umol/L, dGTP 200umol/L, dTTP 200umol/L, dCTP 200umol/L, the upstream primer F10pmol, the downstream primer R10pmol, template DNA0.1ug, Taq DNA polymerase 2.5u, Mg²⁺1.5mmol/L, and the balance of double distilled water and/or triple distilled water;

the reaction conditions of the PCR amplification system are as follows:

denaturation at 94 ℃ for 60 seconds, annealing at 55-60 ℃ for 60 seconds, derivatization at 72 ℃ for 120 seconds, and repeating for 25 cycles in this order.

(ii) Mixing 5 mu g of pPICZ alpha A vector and 50u of BamH I restriction endonuclease to obtain a mixture; and carrying out enzyme digestion on the mixture at 37 ℃ overnight to obtain an enzyme digestion product, and then recovering the enzyme digestion product by adopting agarose nucleic acid electrophoresis to obtain the pPICZ alpha A vector after enzyme digestion.

(iii) And adopting an easy Geno rapid recombinant cloning kit to connect the CUP1 gene fragment with the digested pPICZ alpha A vector to obtain the pCUP1 vector plasmid.

Secondly, constructing the pCUP1-EGFP expression plasmid

(i) Mixing 5 mu g of pCUP1 carrier plasmid, 5u of EcoR I restriction endonuclease and 50u of Not I restriction endonuclease to obtain a mixture; and carrying out enzyme digestion on the mixture at 37 ℃ overnight to obtain an enzyme digestion product, and then recovering the enzyme digestion product by adopting agarose nucleic acid electrophoresis to obtain the pCUP1 vector plasmid after enzyme digestion.

(ii) Amplifying the EGFP gene by adopting a PCR amplification system to obtain an EGFP gene PCR product, and then recovering the EGFP gene PCR product by adopting agarose nucleic acid electrophoresis to obtain an EGFP fragment.

(iii) And adopting an easy Geno rapid recombinant cloning kit to connect the EGFP fragment with the pCUP1 vector plasmid after enzyme digestion to obtain the pCUP1-EGFP expression plasmid.

The sequence of the pCUP1-EGFP expression plasmid is as follows:

thirdly, screening recombinant pichia pastoris with high copy number and foreign gene insertion

(i) Linearizing the pCUP1-EGFP expression plasmid by using Sac I restriction endonuclease to obtain a linearized pCUP1-EGFP expression plasmid, comprising the following steps: (1) mixing said Sac I restriction endonuclease 100u with 10 ug of said pCUP1-EGFP expression plasmid; (2) digesting the mixture obtained in the step (1) at 37 ℃ overnight; (3) reacting the reactant obtained in the step (2) at the temperature of 95 ℃ for 10 minutes, and then stopping the reaction; (4) adding a sodium acetate solution with the concentration of 3M and absolute ethyl alcohol into the reactant obtained in the step (3), centrifuging, and taking a precipitate; the volume ratio of the reactant obtained in the step (3) to the sodium acetate solution is 10: 1.5; the volume ratio of the reactant obtained in the step (3) to the absolute ethyl alcohol is 1: 3; (4) washing the precipitate obtained in the step (3) by using an ethanol solution with the mass concentration of 70%, centrifuging, taking the precipitate, and drying to obtain the linearized pCUP1-EGFP expression plasmid.

(ii) Transforming X33 pichia pastoris competent cells by adopting the linearized pCUP1-EGFP expression plasmid, then coating a YPDS-Zeocin plate, and culturing to obtain a first colony, wherein the method comprises the following steps: (1) mixing 10 μ g of the linearized pCUP1-EGFP expression plasmid with 100 μ L of the Pichia X33 competent cells; (2) keeping the mixture obtained in the step (1) for 5min under an ice bath condition, and then carrying out electric shock; the conditions of the electric shock are as follows: the voltage was 1.5kV, the capacitance was 25. mu.F, the resistance was 400. omega. and the shock time was 10 msec. (3) Adding the reactant obtained in the step (2) into a sorbitol solution with the concentration of 1.5M and the temperature of 4 ℃; (4) spreading the mixture obtained in step (3) on the YPDS-Zeocin plate, and culturing at 30 ℃ until the first colony appears.

(iii) Inoculating the first colony on a first BMGY liquid medium (containing copper sulfate with the concentration of 0.3 mM) to obtain a culture, and inoculating the culture on a second BMGY liquid medium (containing copper sulfate with the concentration of 1 mM) to obtain a bacterial liquid, wherein the method comprises the following steps of: (1) inoculating the first colony to the first BMGY liquid culture medium, and then performing shake culture for 13h at the temperature of 30 ℃ and the speed of 200 rpm; (2) and (2) inoculating the culture obtained in the step (1) to the second BMGY liquid culture medium, performing shaking culture for 25h, centrifuging, and taking the supernatant to obtain the bacterial liquid.

(iv) And (3) resuspending the bacterial liquid by using the third BMGY culture medium (containing copper sulfate with the concentration of 2 mM), coating the BMGY plate (containing copper sulfate with the concentration of 2 mM), and standing and culturing at the temperature of 30 ℃ until monoclonals appear, wherein the grown clones are colonies resistant to copper ions (containing clones with increased copy number of CUP 1), and thus obtaining the recombinant pichia pastoris with high copy number inserted with the foreign gene.

(v) And (3) selecting the third BMGY culture medium with the monoclonal appeared, carrying out shake culture until OD600 reaches 1-2, centrifuging, replacing with a BMMY culture medium, inducing for 48-72 hours, supplementing methanol every 24 hours, keeping the final concentration of methanol at 0.5%, and finishing the induced expression of the foreign protein.

Example 3: fluorescent quantitative RT _ PCR detection

Aiming at the recombinant pichia pastoris high-copy single colony obtained by the screening method in the embodiment 1-2, a fluorescent quantitative RT _ PCR method is adopted for detection;

(1) selecting 30 recombinant pichia pastoris high-copy single colonies obtained by the screening method, extracting yeast genome DNA by adopting a commercial yeast genome DNA extraction kit, determining OD280 and OD260, taking 20 groups of samples with DNA quality close to each other, and diluting to the same concentration to obtain a sample to be detected;

(2) and detecting the sample to be detected by adopting a fluorescent quantitative RT _ PCR method.

Wherein, the primer is:

EGFP-L：5'AAGGGCATCGACTTCAAGGA；

EGFP-R：5'GGTGTTCTGCTGGTAGTGGT；

in the fluorescent quantitative RT _ PCR method, the reaction system is 25ul, which includes: 2 x amplification buffer 12.5ul, primer EGFP-L (10 uM concentration) 10ul, primer EGFP-R (10 uM concentration) 10ul, polymerase 0.5u, template DNA100pg-100ng, and the balance deionized water.

The reaction conditions are as follows: sequentially modifying at 95 ℃ for 5min, at 95 ℃ for 10 s, annealing at 60 ℃ for 45 s, and repeating for 30-40 cycles; wherein the dissolution curve analysis conditions are as follows: in this order at a temperature of 95 ℃ for 15 seconds, at a temperature of 60 ℃ for 1min, at a temperature of 95 ℃ for 15 seconds, and at a temperature of 60 ℃ for 15 seconds.

(3) The detection result shows that in 20 groups of samples to be detected, the average CT values of the colonies screened by the copper ions are respectively from small to large (the larger the copy number is, the smaller the CT value is): 16.75, 17.45, 19.36, 25.53, 27.96, 28.37, 28.54, 29.04, 29.61, 29.77, 30.6, 36.11, 36.13, 36.25, 36.26, 36.47, 36.51, 36.95, 37.12, 37.49; as described in the screening method of the invention, under the induction action of copper ions, high-copy foreign gene insertion which is spontaneously formed by pichia pastoris can occur, the copy number of foreign gene (EGFP) expression which is positioned near a CUP1 locus is amplified together, and high-copy-number clones can be screened out under the high copper ion concentration.

In addition, 30 single colonies obtained by the prior art and subjected to pPICZ alpha-EGFP/x 33 Zeocin screening are picked, yeast genome DNA is extracted by adopting a commercial yeast genome DNA extraction kit, OD280 and OD260 are measured, 20 samples with DNA quality close to each other are taken, and the samples are diluted to the same concentration to obtain a comparison sample to be measured. The contrast sample to be detected is detected by adopting the fluorescent quantitative RT _ PCR method, and the detection result shows that the average CT value of the bacterial colony screened by the copper ions in 20 groups of contrast samples to be detected is respectively as follows from small to large: 24.18, 26.57, 27.36, 27.63, 27.95, 28.06, 28.35, 28.36, 28.61, 28.87, 29.33, 29.34, 29.4, 29.62, 29.91, 29.94, 30.25, 35.62, 36.03, 36.42.

As shown in fig. 3, the detection results of the present example show that:

the difference between the clones of the single colony obtained by the prior art after the pPICZ alpha-EGFP/x 33 Zeocin screening is small, while the difference between the clones screened by the copper ions in the screening method is relatively large;

in the screening method, copper ions can promote the CUP1 gene to generate copy number mutation, and forward mutation and reverse mutation exist; under the induction action of copper ions, high-copy foreign gene insertion which is spontaneously formed by pichia pastoris can occur, the copy number of EGFP expression which is positioned near a CUP1 locus is amplified together, and the clone with high copy number can be screened out under the high copper ion concentration.

In conclusion, the above description of the embodiments of the present invention is not intended to limit the present invention, and those skilled in the art can make various changes or modifications according to the present invention without departing from the spirit of the present invention, which falls within the scope of the appended claims.

Sequence listing

<110> Wuhan Huamei bioengineering Co., Ltd

<120> construction and application of pCUP1 carrier plasmid

<141> 2020-02-28

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5580

<212> DNA

<213> Artificial Sequence

<400> 1

agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60

gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120

tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180

agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240

acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 300

tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360

agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420

gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480

ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 540

cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600

ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660

ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720

gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 780

atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 840

actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 900

caacttgaga agatcaaaaa acaactaatt attcgaaacg atgagatttc cttcaatttt 960

tactgctgtt ttattcgcag catcctccgc attagctgct ccagtcaaca ctacaacaga 1020

agatgaaacg gcacaaattc cggctgaagc tgtcatcggt tactcagatt tagaagggga 1080

tttcgatgtt gctgttttgc cattttccaa cagcacaaat aacgggttat tgtttataaa 1140

tactactatt gccagcattg ctgctaaaga agaaggggta tctctcgaga aaagagaggc 1200

tgaagctgaa ttcacgtggc ccagccggcc gtctcggatc ggtacctcga gccgcggcgg 1260

ccgccagctt tctagaacaa aaactcatct cagaagagga tctgaatagc gccgtcgacc 1320

atcatcatca tcatcattga gtttgtagcc ttagacatga ctgttcctca gttcaagttg 1380

ggcacttacg agaagaccgg tcttgctaga ttctaatcaa gaggatgtca gaatgccatt 1440

tgcctgagag atgcaggctt catttttgat acttttttat ttgtaaccta tatagtatag 1500

gatttttttt gtcattttgt ttcttctcgt acgagcttgc tcctgatcag cctatctcgc 1560

agctgatgaa tatcttgtgg taggggtttg ggaaaatcat tcgagtttga tgtttttctt 1620

ggtatttccc actcctcttc agagtacaga agattaagtg agaccttcgt ttgtgcgcaa 1680

tagaggcagg tatcggagat aggttttcag cagcgggtac catgaatagc tttttttttt 1740

ttttttattc gaaatctggg gattctatac agagttgtaa gttaggcaaa ctagaatttg 1800

gtaataatat tttattcttg gggcgacata tggagatact ttatttcctt ttcttaatta 1860

ttaacgtata cctataaatt aacaaagtat ctaaacaaaa tacataagtg tactcaaact 1920

gagtagaatc gtcgattaaa cttccttctc cttttaaaaa ttaaaaacag caaatagtta 1980

gatgaatata ttaaagacta ttcgtttcat ttcccagagc agcatgactt cttggtttct 2040

tcagacttgt taccgcaggg gcatttgtcg tcgctgttac accccgttgg gcagctacat 2100

gatttttggc attgttcatt atttttgcag ctaccacatt ggcattggca ctcatgacct 2160

tcattttgga agttaattaa ttcgctgaac attttatgtg atgattgatt gattgattgt 2220

acagtttgtt tttcttaata tctatttcga tgacttctat atgatattgc actaacaaga 2280

agatattata atgcaattga tacaagacaa ggagttattt gcttctcttt tatatgattc 2340

tgacaatcca tattgcgttg gtagtctttt ttgctggaac ggttcagcgg aaaagacgca 2400

tcgctctttt tgcttctaga agaaatgcca gcaaaagaat ctcttgacag tgactgacag 2460

caaaaatgtc tttttctaac tagtaacaag gctaagatat cagcctgaaa taaagggtgg 2520

tgaagtaata attaaatcat ccgtataaac ctatacacat atatgaggaa aaataataca 2580

aaagtgtttt aaatacagat acatacatga acatatgcac gtatagcgcc caaatgtcgg 2640

taatgggatc ggcttactaa ttataaaatg catcatagaa atcgttgaag tttgccgtag 2700

taatacccag attatcagat tccaaatcct tgtcaataat tatactcctt tggacaactt 2760

ctctttccat taaaaaatct gaaatctcct taaattttaa atagattctg ttcagttcac 2820

taacggggaa tttcaagaga acatttttgt tcttcgccga ctgactataa tctgtaacat 2880

tattgttatc agagtttctc gcaaaatttt gttttttctt gctaaatctc agcatatatt 2940

taatcagatt caaaaccttg ttgaaacctt taatagattt gaaacttccg ttgctattca 3000

tttcatctcg taaaaaggat acgataattt ctattttttt taaaatttcc aaaatcttgt 3060

catgaatcaa tagcaattga acattaatct cctcatttga aagatttttg taaaattcgt 3120

catataatat tacttcacaa cgttggaaaa tagcaaatgt gattgctata aaattctgta 3180

agatttcaat aaaatgattt gcgaataaaa attctttacc attagaatga aagcgattat 3240

tgccgcttga aaatgacttt atcgacttta tggggaagat aaaattaaat gttattgagt 3300

aaaaaatgtg catattagaa ataattttca tcagatcctt tgcacatctt tcagagttcg 3360

aggtcttatt gttgttagaa gaatgttgaa ctgccatgga caaagaggat tcgttttgaa 3420

caaaaaggaa aaaatttgta taaacaatgg tattgataaa atttaaagtg tctttccatt 3480

cttttctgac ttcgttgtca tgaaaatata agtctactgt attactcacg cccatagtca 3540

aggtttctaa cagactttca attttggtta aatttactgg caagtagaaa ggaacacctt 3600

gcagaatatt tatcaatttt gcttgcgttt ccagtaattt taaatcgtta gcaattaaag 3660

gaatgatccc ccacacacca tagcttcaaa atgtttctac tcctttttta ctcttccaga 3720

ttttctcgga ctccgcgcat cgccgtacca cttcaaaaca cccaagcaca gcatactaaa 3780

ttttccctct ttcttcctct agggtgtcgt taattacccg tactaaaggt ttggaaaaga 3840

aaaaagagac cgcctcgttt ctttttcttc gtcgaaaaag gcaataaaaa tttttatcac 3900

gtttcttttt cttgaaattt ttttttttag tttttttctc tttcagtgac ctccattgat 3960

atttaagtta ataaacggtc ttcaatttct caagtttcag tttcattttt cttgttctat 4020

tacaactttt tttacttctt gttcattaga aagaaagcat agcaatctaa tctaaggggc 4080

ggtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg 4140

aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc 4200

ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac 4260

ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg 4320

gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag 4380

tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc 4440

ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac 4500

ttcgtggccg aggagcagga ctgacacgtc cgacggcggc ccacgggtcc caggcctcgg 4560

agatccgtcc cccttttcct ttgtcgatat catgtaatta gttatgtcac gcttacattc 4620

acgccctccc cccacatccg ctctaaccga aaaggaagga gttagacaac ctgaagtcta 4680

ggtccctatt tattttttta tagttatgtt agtattaaga acgttattta tatttcaaat 4740

ttttcttttt tttctgtaca gacgcgtgta cgcatgtaac attatactga aaaccttgct 4800

tgagaaggtt ttgggacgct cgaaggcttt aatttgcaag ctggagacca acatgtgagc 4860

aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 4920

gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 4980

gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 5040

tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 5100

ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 5160

ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 5220

tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 5280

tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 5340

ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 5400

aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 5460

ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 5520

tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatc 5580

<210> 2

<211> 6249

<212> DNA

<213> Artificial Sequence

<400> 2

agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60

gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120

tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180

agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240

acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 300

tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360

agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420

gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480

ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 540

cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600

ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660

ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720

gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 780

atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 840

actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 900

caacttgaga agatcaaaaa acaactaatt attcgaaacg atgagatttc cttcaatttt 960

tactgctgtt ttattcgcag catcctccgc attagctgct ccagtcaaca ctacaacaga 1020

agatgaaacg gcacaaattc cggctgaagc tgtcatcggt tactcagatt tagaagggga 1080

tttcgatgtt gctgttttgc cattttccaa cagcacaaat aacgggttat tgtttataaa 1140

tactactatt gccagcattg ctgctaaaga agaaggggta tctctcgaga aaagagaggc 1200

tgaagctatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga 1260

gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc 1320

cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg 1380

gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca 1440

catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac 1500

catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga 1560

caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct 1620

ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca 1680

gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca 1740

gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga 1800

caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca 1860

catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta 1920

caagtaaggc cgccagcttt ctagaacaaa aactcatctc agaagaggat ctgaatagcg 1980

ccgtcgacca tcatcatcat catcattgag tttgtagcct tagacatgac tgttcctcag 2040

ttcaagttgg gcacttacga gaagaccggt cttgctagat tctaatcaag aggatgtcag 2100

aatgccattt gcctgagaga tgcaggcttc atttttgata cttttttatt tgtaacctat 2160

atagtatagg attttttttg tcattttgtt tcttctcgta cgagcttgct cctgatcagc 2220

ctatctcgca gctgatgaat atcttgtggt aggggtttgg gaaaatcatt cgagtttgat 2280

gtttttcttg gtatttccca ctcctcttca gagtacagaa gattaagtga gaccttcgtt 2340

tgtgcgcaat agaggcaggt atcggagata ggttttcagc agcgggtacc atgaatagct 2400

tttttttttt tttttattcg aaatctgggg attctataca gagttgtaag ttaggcaaac 2460

tagaatttgg taataatatt ttattcttgg ggcgacatat ggagatactt tatttccttt 2520

tcttaattat taacgtatac ctataaatta acaaagtatc taaacaaaat acataagtgt 2580

actcaaactg agtagaatcg tcgattaaac ttccttctcc ttttaaaaat taaaaacagc 2640

aaatagttag atgaatatat taaagactat tcgtttcatt tcccagagca gcatgacttc 2700

ttggtttctt cagacttgtt accgcagggg catttgtcgt cgctgttaca ccccgttggg 2760

cagctacatg atttttggca ttgttcatta tttttgcagc taccacattg gcattggcac 2820

tcatgacctt cattttggaa gttaattaat tcgctgaaca ttttatgtga tgattgattg 2880

attgattgta cagtttgttt ttcttaatat ctatttcgat gacttctata tgatattgca 2940

ctaacaagaa gatattataa tgcaattgat acaagacaag gagttatttg cttctctttt 3000

atatgattct gacaatccat attgcgttgg tagtcttttt tgctggaacg gttcagcgga 3060

aaagacgcat cgctcttttt gcttctagaa gaaatgccag caaaagaatc tcttgacagt 3120

gactgacagc aaaaatgtct ttttctaact agtaacaagg ctaagatatc agcctgaaat 3180

aaagggtggt gaagtaataa ttaaatcatc cgtataaacc tatacacata tatgaggaaa 3240

aataatacaa aagtgtttta aatacagata catacatgaa catatgcacg tatagcgccc 3300

aaatgtcggt aatgggatcg gcttactaat tataaaatgc atcatagaaa tcgttgaagt 3360

ttgccgtagt aatacccaga ttatcagatt ccaaatcctt gtcaataatt atactccttt 3420

ggacaacttc tctttccatt aaaaaatctg aaatctcctt aaattttaaa tagattctgt 3480

tcagttcact aacggggaat ttcaagagaa catttttgtt cttcgccgac tgactataat 3540

ctgtaacatt attgttatca gagtttctcg caaaattttg ttttttcttg ctaaatctca 3600

gcatatattt aatcagattc aaaaccttgt tgaaaccttt aatagatttg aaacttccgt 3660

tgctattcat ttcatctcgt aaaaaggata cgataatttc tatttttttt aaaatttcca 3720

aaatcttgtc atgaatcaat agcaattgaa cattaatctc ctcatttgaa agatttttgt 3780

aaaattcgtc atataatatt acttcacaac gttggaaaat agcaaatgtg attgctataa 3840

aattctgtaa gatttcaata aaatgatttg cgaataaaaa ttctttacca ttagaatgaa 3900

agcgattatt gccgcttgaa aatgacttta tcgactttat ggggaagata aaattaaatg 3960

ttattgagta aaaaatgtgc atattagaaa taattttcat cagatccttt gcacatcttt 4020

cagagttcga ggtcttattg ttgttagaag aatgttgaac tgccatggac aaagaggatt 4080

cgttttgaac aaaaaggaaa aaatttgtat aaacaatggt attgataaaa tttaaagtgt 4140

ctttccattc ttttctgact tcgttgtcat gaaaatataa gtctactgta ttactcacgc 4200

ccatagtcaa ggtttctaac agactttcaa ttttggttaa atttactggc aagtagaaag 4260

gaacaccttg cagaatattt atcaattttg cttgcgtttc cagtaatttt aaatcgttag 4320

caattaaagg aatgatcccc cacacaccat agcttcaaaa tgtttctact ccttttttac 4380

tcttccagat tttctcggac tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag 4440

catactaaat tttccctctt tcttcctcta gggtgtcgtt aattacccgt actaaaggtt 4500

tggaaaagaa aaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat 4560

ttttatcacg tttctttttc ttgaaatttt tttttttagt ttttttctct ttcagtgacc 4620

tccattgata tttaagttaa taaacggtct tcaatttctc aagtttcagt ttcatttttc 4680

ttgttctatt acaacttttt ttacttcttg ttcattagaa agaaagcata gcaatctaat 4740

ctaaggggcg gtgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac 4800

aaggtgagga actaaaccat ggccaagttg accagtgccg ttccggtgct caccgcgcgc 4860

gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg 4920

gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag 4980

gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg 5040

tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg 5100

accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac 5160

tgcgtgcact tcgtggccga ggagcaggac tgacacgtcc gacggcggcc cacgggtccc 5220

aggcctcgga gatccgtccc ccttttcctt tgtcgatatc atgtaattag ttatgtcacg 5280

cttacattca cgccctcccc ccacatccgc tctaaccgaa aaggaaggag ttagacaacc 5340

tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa cgttatttat 5400

atttcaaatt tttctttttt ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa 5460

aaccttgctt gagaaggttt tgggacgctc gaaggcttta atttgcaagc tggagaccaa 5520

catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 5580

tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 5640

gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 5700

ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 5760

cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 5820

caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 5880

ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 5940

taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 6000

taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 6060

cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 6120

tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 6180

gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 6240

catgagatc 6249

<210> 3

<211> 717

<212> DNA

<213> Artificial Sequence

<400> 3

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaag 717

Claims (1)

1. A screening method of recombinant pichia pastoris with high copy and stable insertion of exogenous genes is characterized by comprising the following steps:

carrying out double enzyme digestion on the pCUP1 vector plasmid by using EcoRI restriction endonuclease and NotI restriction endonuclease to obtain an enzyme digestion product, and then recovering the enzyme digestion product by using electrophoresis to obtain the pCUP1 vector plasmid after enzyme digestion;

adopting a PCR amplification system to amplify the exogenous gene to obtain an exogenous gene PCR product, and then adopting electrophoresis to recover the exogenous gene PCR product to obtain an exogenous gene fragment;

connecting the exogenous gene fragment with the pCUP1 vector plasmid after enzyme digestion to obtain pCUP 1-exogenous gene expression plasmid;

linearizing the pCUP 1-exogenous gene expression plasmid by adopting Sac I restriction endonuclease to obtain a linearized pCUP 1-exogenous gene expression plasmid;

transforming X33 pichia pastoris competent cells by adopting the linearized pCUP 1-exogenous gene expression plasmid, then coating a YPDS-Zeocin plate, and culturing to obtain a first colony;

inoculating the first colony to a first BMGY liquid culture medium to obtain a culture, and inoculating the culture to a second BMGY liquid culture medium to obtain a bacterial liquid;

re-suspending the bacterium solution by using a third BMGY culture medium, coating a third BMGY flat plate, and culturing to obtain a recombinant pichia pastoris high-copy bacterial colony;

wherein the content of the first and second substances,

the YPDS-Zeocin flat plate comprises: 1% yeast extract, 2% peptone, 2% glucose, 1mol/L sorbitol, 2% agar, 100. mu.g/ml Zeocin;

the first BMGY liquid culture medium comprises: 1% yeast extract, 2% peptone, 100mM potassium dihydrogen phosphate, pH 6.0, 1.34% YNB, 4X10^-5% biotin, glycerol at a concentration of 1%, copper sulfate at a concentration of 0.3 mM;

the second BMGY liquid culture medium comprises: 1% yeast extract, 2% peptone, 100mM potassium dihydrogen phosphate, pH 6.0, 1.34% YNB, 4X10^-5% biotin, glycerol at a concentration of 1%, copper sulfate at a concentration of 1 mM;

the third BMGY culture medium comprises: 1% yeast extract, 2% peptone, 100mM potassium dihydrogen phosphate, pH 6.0, 1.34% YNB, 4X10^-5% biotin, glycerol at a concentration of 1%, copper sulfate at a concentration of 2 mM;

the third BMGY flat plate comprises: 1% yeast extract, 2% peptone, 1.5% agarose, 100mM potassium dihydrogen phosphate, pH 6.0, 1.34% YNB, 4X10^-5% biotin, glycerol at a concentration of 1%, copper sulfate at a concentration of 2 mM;

the construction method of the pCUP1 vector plasmid is as follows:

amplifying a CUP1 gene in the saccharomyces cerevisiae by adopting a PCR amplification system to obtain a PCR product, and recovering the PCR product by adopting electrophoresis to obtain a CUP1 gene fragment;

carrying out enzyme digestion on the pPICZ alpha A vector by using BamH I restriction endonuclease to obtain an enzyme digestion product, and recovering the enzyme digestion product by using electrophoresis to obtain the enzyme digested pPICZ alpha A vector;

connecting the CUP1 gene segment with the digested pPICZ alpha A vector to obtain the pCUP1 vector plasmid;

wherein the content of the first and second substances,

the amplification primers in the PCR amplification system are as follows:

an upstream primer F: at the time of the start of the 5' TGAGACCTTCGTTTGTGCGcaatagaggcaggtatc,

a downstream primer R: 5' ATGGTGTGTGGGGGATCattcctttaattgctaa;

the PCR amplification system is 100ul, and comprises:

10 Xamplification buffer solution 10ul, dATP 200umol/L, dGTP 200umol/L, dTTP 200umol/L, dCTP 200umol/L, the upstream primer F10-100 pmol, the downstream primer R10-100 pmol, template DNA 0.1-2 ug, Taq DNA polymerase 2.5u, Mg²⁺1.5mmol/L, and the balance of double distilled water and/or triple distilled water;

the reaction conditions of the PCR amplification system are as follows:

sequentially denaturing at 94 ℃ for 60 seconds, annealing at 55-60 ℃ for 60 seconds, derivatizing at 72 ℃ for 120 seconds, and repeating for 25-30 cycles.

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