CN114317303A - Recombinant pichia pastoris for secretory expression of recombinant stinkbug defensin THA and construction and expression methods thereof - Google Patents

Abstract

The invention discloses a recombinant pichia pastoris for secretory expression of recombinant stinkbug defensin THA and a construction and expression method thereof. The recombinant pichia pastoris is obtained by inserting the THA expression cassette into two different sites of a genome to construct, the genetic property is stable, the THA expression quantity is high, and the THA expression quantity can reach more than 1.5g/L through detection and is more than twice of that of the prior art.

Description

Recombinant pichia pastoris for secretory expression of recombinant stinkbug defensin THA and construction and expression methods thereof

Technical Field

The invention relates to the field of genetic engineering, and particularly relates to recombinant pichia pastoris for secretory expression of recombinant stinkbug defensin THA and a construction method thereof.

Background

The stink bug defensin thanatin (tha) is an antibacterial peptide which is found by french scientists in haemolymph in vivo of hemiptera insect lucilia punctifera (podius maculiventris) 24 hours after infection by escherichia coli or micrococcus luteus in 1996 and has antibacterial activity on certain gram-negative bacteria, gram-positive bacteria and fungi. Mature THA consists of 21 amino acids with the sequence GSKKPVPIIYCNRRTGKCQRM. THA contains in solution a steric structure formed by two antiparallel beta-sheets, which steric structure is stabilized by a disulfide bond formed by cysteines at positions 11 and 18 (Cys11-Cys 18). Research shows that THA has strong bacteriostatic activity on escherichia coli, salmonella, micrococcus luteus, candida albicans and the like. As an antibacterial substance, THA has potential application value in the fields of medicine, food, feed and the like.

For practical application of THA, large-scale production methods must be established. THA can be obtained in three ways: biological extraction, chemical synthesis and biological synthesis. THA produced by the adelphocoris suturalis is naturally very little, so that it is not feasible to produce THA by an extraction method. THA is obtained by a chemical synthesis mode, and the production cost is overhigh at present. Biosynthesis is mainly carried out by constructing an engineering strain capable of heterologously expressing THA through a genetic engineering means and then carrying out mass production through fermentation on an industrial scale. Pichia pastoris has been used for the industrial production of a variety of recombinant polypeptides and proteins due to its ease of culture, strong secretion capacity and low content of contaminating proteins in the secreted product. An engineering bacterium (CN 107904182A) which is obtained by inserting a recombinant THA expression cassette into a pichia pastoris alcohol oxidase gene site is constructed in the earlier stage of Liangweifan and the like, but the fermentation expression level is not high, and the THA content is 0.6 g/L. On the basis of engineering bacteria with a recombinant THA expression cassette inserted in a single site, the THA secretion expression level of the engineering bacteria (CN 108342334A) obtained by ultraviolet mutagenesis is improved (the THA content reaches 0.76g/L), but the expression level is still not high, and the genetic characters of the engineering bacteria are found to be unstable in the subsequent fermentation production process.

It is seen that improvements and enhancements to the prior art are needed.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide the recombinant pichia pastoris for secretory expression of the recombinant stinkbug defensin THA and the construction method thereof, and aims to solve the problems of low expression quantity and unstable genetic character of the recombinant pichia pastoris for stinkbug defensin THA in the prior art.

In order to achieve the purpose, the invention adopts the following technical scheme:

a recombinant Pichia pastoris for secreting and expressing recombinant stink bug defensin THA, wherein the preservation number of the recombinant Pichia pastoris (Pichia pastoris) is GDMCC No. 61977.

A method for constructing recombinant pichia pastoris is used for constructing the recombinant pichia pastoris for secreting and expressing the recombinant stinkbug defensin THA, and comprises the following steps:

step S001, designing a recombinant stink bug defensin THA coding gene: using a THA mature peptide sequence as a target product, and designing a THA coding gene according to pichia pastoris codon preference;

step S002, constructing an expression vector: respectively connecting the THA coding gene designed in the step S001 to vectors pPICZ alpha A and pPIC3.5K, and respectively constructing expression vectors pPICZ alpha A-THA and pPIC3.5K-THA;

step S003, constructing recombinant pichia pastoris: respectively carrying out linearization treatment on the pPICZ alpha A-THA and the pPIC3.5K-THA expression vectors, then transferring the pPICZ alpha A-THA linearization expression vectors into pichia pastoris competent cells, inoculating the converted pichia pastoris into a screening culture medium for culture, detecting the strain expression quantity after obtaining single strains, and selecting an optimal strain THA-1; and then transferring the pPIC3.5K-THA linear expression vector into a strain THA-1 competent cell, performing strain expression amount detection again after obtaining single strains through screening culture medium culture, and selecting an optimal strain THA-2 to obtain the recombinant pichia pastoris for secreting and expressing the recombinant stinkbug defensin THA.

The method for constructing the recombinant pichia pastoris is characterized in that in the step S001, the THA coding gene sequence is SEQ ID No. 2.

The method for constructing the recombinant pichia pastoris is characterized in that in the step S002, the construction of the pPICZ alpha A-THA expression vector comprises the following steps: adding an XhoI sequence at the 5 'end of the THA coding gene sequence, adding an XbaI sequence at the 3' end, performing double digestion treatment by using restriction enzymes XhoI and XbaI, and connecting the THA coding gene subjected to double digestion treatment to a linearized pPICZ alpha A vector which is also subjected to double digestion treatment by using XhoI and XbaI by using T4DNA ligase to construct the pPICZ alpha A-THA expression vector.

The method for constructing the recombinant pichia pastoris is characterized in that in the step S002, the construction of the pPIC3.5K-THA expression vector comprises the following steps: adding an alpha-factor secretion signal peptide sequence at the 5 'end of a THA coding gene sequence, then adding a BamHI sequence in front of an initiation codon of the signal peptide, adding a NotI sequence at the 3' end of the THA coding gene sequence to obtain a fusion sequence, then carrying out double digestion treatment by using restriction enzymes BamHI and NotI, and then connecting the THA coding gene subjected to double digestion treatment to a linearized pPIC3.5K vector which is also subjected to double digestion treatment by using BamHI and NotI by using T4DNA ligase to construct the pPIC3.5K-THA expression vector.

In the step S003, the pPICZ alpha A-THA and pPIC3.5K-THA linearized expression vector is transferred into a competent cell by electrotransformation.

In the step S003, the pPICZ α a-THA expression vector is linearized with a PmeI restriction enzyme.

In the method for constructing the recombinant pichia pastoris, in the step S003, the pPIC3.5K-THA expression vector is subjected to linearization treatment by SalI restriction endonuclease.

The method for constructing the recombinant pichia pastoris is characterized in that in the step S003, the detection of the strain expression level comprises the following steps: and (3) selecting a single colony for culturing, inducing the single colony for 48 hours by using 1% methanol after the single colony is stabilized, collecting fermentation supernatant, centrifuging the supernatant, detecting the size of a bacteriostatic zone, and selecting a strain with the optimal expression quantity.

An inducible expression method of recombinant pichia pastoris is used for the recombinant pichia pastoris to secrete and express recombinant stinkbug defensin THA, and comprises the following steps:

step S101, primary culture: inoculating the recombinant pichia pastoris into 25mL of BMGY medium, and culturing for 24 hours at 30 ℃ and 220rpm to obtain a first-stage seed solution;

step S102, secondary culture: inoculating 20mL of the first-stage seed solution into 200mL of BMGY culture medium, and culturing at 30 ℃ and 220rpm for 24 hours to obtain a second-stage seed solution;

step S103, three-stage culture: inoculating the second-stage seed liquid into a fermentation tank containing 2L BSM culture medium, and culturing at 29.5-30.5 deg.C, 19.5-20.5% dissolved oxygen and 4.5-5.5% pH;

step S104, induced expression: when the glycerol in the BSM culture medium is exhausted, 10% of glycerol is fed, after the glycerol is exhausted, the dissolved oxygen rises to 100%, starving is carried out for 1 hour, then methanol is fed to induce THA expression, the induction time is 96 hours, the temperature is controlled to be 25.5-26.5 ℃, and the pH is controlled to be 4-4.5.

Has the advantages that:

the invention provides a recombinant pichia pastoris for secretory expression of recombinant stink bug defensin THA, which has stable genetic performance and high THA expression quantity, can reach more than 1.5g/L through detection, is more than twice of that of the prior art, and lays a good foundation for large-scale production of THA antibacterial peptide.

In addition, the invention also provides a construction method of the recombinant pichia pastoris, which is used for constructing the recombinant pichia pastoris for secreting and expressing the recombinant stinkbug defensin THA. In addition, the copy number of the THA coding gene in pichia pastoris is increased by adopting a multi-site insertion mode, and the expression quantity of the recombinant pichia pastoris THA is improved.

Drawings

FIG. 1 is a scheme showing the construction of pPICZ. alpha.A-THA expression vector.

FIG. 2 is a scheme showing the construction of pPIC3.5K-THA expression vector.

FIG. 3 shows the results of the test of the bacteriostatic activity of the fermentation supernatant of the selected strain in step S003 against Escherichia coli ATCC 25922.

FIG. 4 shows Tricine-SDS-PAGE protein electrophoresis results of the fermentation supernatants of THA-1 and THA-2 strains.

FIG. 5 shows reversed-phase HPLC results of fermentation supernatants of THA-1 and THA-2 strains.

FIG. 6 shows the mass spectra of THA purified from the fermentation supernatant by reverse phase HPLC.

FIG. 7 shows the results of the measurement of the bacteriostatic activity of the fermentation supernatants of the strains THA-1 and THA-2 against E.coli ATCC25922 after the scale-up culture.

Detailed Description

The invention provides a recombinant pichia pastoris for secretory expression of recombinant stinkbug defensin THA and a construction method thereof, and in order to make the purpose, technical scheme and effect of the invention clearer and more clear and definite, the invention is further described in detail with reference to the attached drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

A method for constructing recombinant pichia pastoris, which is used for constructing the recombinant pichia pastoris for secreting and expressing recombinant stinkbug defensin THA, comprises the following steps:

step S001, designing a recombinant stink bug defensin THA coding gene: THA mature peptide sequence SEQ ID NO.1 is used as a target product, and a THA coding gene is designed according to pichia pastoris codon preference; the THA coding gene obtained by design is SEQ ID NO. 2.

Step S002, constructing an expression vector: respectively connecting the THA coding gene designed in the step S001 to vectors pPICZ alpha A and pPIC3.5K, and respectively constructing expression vectors pPICZ alpha A-THA and pPIC3.5K-THA; the integration site of the pPICZ alpha A vector on the pichia pastoris genome is AOX1, the integration site of the pPIC3.5K vector on the pichia pastoris genome is HIS4, and the multi-site integration can reduce the loss of exogenous genes during strain passage, so that the genetic stability of the strain is improved.

Referring to FIG. 1, the construction of pPICZ α A-THA expression vector includes the following steps: adding an XhoI sequence at the 5 'end of the THA coding gene sequence, adding an XbaI sequence at the 3' end, performing double enzyme digestion treatment by using restriction enzymes XhoI and XbaI, and connecting the THA coding gene subjected to double enzyme digestion treatment to a linearized pPICZ alpha A vector subjected to double enzyme digestion treatment by using the XhoI and XbaI by using T4DNA ligase to construct the pPICZ alpha A-THA expression vector;

validation of pPICZ α A-THA expression vector: the ligation products were heat shock transformed into E.coli Top10 competent cells at 42 ℃ and then E.coli Top10 was spread on a bleomycin-containing LB resistant plate and cultured overnight at 37 ℃ until single colonies grew. Single colonies were selected for colony PCR and positive transformants verified. The verification primer is as follows: 5 AOX-F: 5'-GACTGGTTCCAATTGACAAGC-3' and 3 AOX-R: 5'-GCAAATGGCATTCTGACATCC-3' are provided. And (4) selecting positive transformants for sequencing, and storing the correct strains in glycerin tubes.

Referring to FIG. 2, the construction of pPIC3.5K-THA expression vector includes the following steps: adding an alpha-factor secretion signal peptide sequence (sequence 3) at the 5 'end of a THA coding gene sequence, then adding a BamHI sequence in front of an initiation codon of the signal peptide, adding a NotI sequence at the 3' end of the THA coding gene sequence to obtain a fusion sequence, then carrying out double enzyme digestion treatment on the fusion sequence by using restriction enzymes BamHI and NotI, and then connecting the double enzyme digestion treated fusion sequence to a linearized pPIC3.5K vector which is also subjected to double enzyme digestion treatment by using BamHI and NotI by using T4DNA ligase to construct the pPIC3.5K-THA expression vector; the alpha-factor secretion signal peptide sequence is used for leading THA to be secreted outside a yeast body after being synthesized, so that the THA can be conveniently purified; and an alpha-factor secretion signal peptide sequence is designed in the pPICZ alpha A vector, so that the introduction is not needed.

Validation of pPIC3.5K-THA expression vector: the ligation products were heat-shocked at 42 ℃ into E.coli Top10 competent cells, and then E.coli Top10 was spread on an LB resistant plate containing kanamycin and cultured overnight at 37 ℃ until single colonies grew. Single colonies were selected for colony PCR and positive transformants verified. The verification primer is as follows: 5 AOX-F: 5'-GACTGGTTCCAATTGACAAGC-3' and 3 AOX-R: 5'-GCAAATGGCATTCTGACATCC-3' are provided. And (4) selecting positive transformants for sequencing, and storing the correct strains in glycerin tubes.

Step S003, constructing recombinant pichia pastoris: firstly, PmeI restriction endonuclease is adopted to carry out linearization treatment on a pPICZ alpha A-THA expression vector, electrophoretic separation is carried out, the linearized expression vector is recovered by gel cutting, then the pPICZ alpha A-THA linearized expression vector is electrically transferred to pichia pastoris competent cells, the voltage is 1.5-2.5kV, and host pichia pastoris can select X33 pichia pastoris or other pichia pastoris, such as GS115, SMD1168 and the like; then inoculating the transformed pichia pastoris into a screening culture medium for culture, detecting the strain expression quantity after obtaining monosomyl lag, and selecting an optimal strain THA-1;

and then, carrying out linearization treatment on the pPIC3.5K-THA expression vector by adopting SalI restriction endonuclease, carrying out electrophoretic separation, cutting gel and recovering the linearized expression vector, then transferring the pPIC3.5K-THA linearized expression vector into strain THA-1 competent cells, carrying out strain expression quantity detection again after obtaining single strain by screening culture medium culture, selecting an optimal strain THA-2, and obtaining the recombinant pichia pastoris secreting and expressing the recombinant stinkbug defensin THA.

In step S003, the detection of the expression level of the strain comprises the following steps: and (3) selecting a single colony for culturing, inducing the single colony for 48 hours by using 1% methanol after the single colony is stabilized, collecting fermentation supernatant, centrifuging the supernatant for detecting the size of a bacteriostatic zone, and selecting a strain with the optimal THA expression quantity.

FIG. 3 shows the results of the tests of the bacteriostatic activity of the strain fermentation broth on Escherichia coli ATCC25922, wherein "X33" is a blank strain control, "F1-1" and "F1-2" are THA-1 strains, "F2-1", "F2-2" and "F2-3" are THA-2 strains, and it can be seen from the figure that, in the order of the size of the zone of inhibition, the THA-2 strain > THA-1 strain > blank strain, and after two conversions, the bacteriostatic activity of the THA-2 strain fermentation broth is significantly improved, which means that the expression level of THA is further increased.

The induction expression method of the recombinant pichia pastoris comprises the following steps:

step S101, primary culture: inoculating the recombinant pichia pastoris into 25mL of BMGY medium, and culturing for 24 hours at 30 ℃ and 220rpm to obtain a first-stage seed solution;

step S102, secondary culture: inoculating 20mL of the first-stage seed solution into 200mL of BMGY culture medium, and culturing at 30 ℃ and 220rpm for 24 hours to obtain a second-stage seed solution;

step S103, three-stage culture: inoculating the second-stage seed liquid into a fermentation tank containing 2L BSM culture medium, and culturing at 29.5-30.5 deg.C, 19.5-20.5% dissolved oxygen and 4.5-5.5% pH;

step S104, induced expression: when the glycerol in the BSM culture medium is exhausted, 10% of glycerol is fed, after the glycerol is exhausted, the dissolved oxygen rises to 100%, starving is carried out for 1 hour, then methanol is fed to induce THA expression, the induction time is 96 hours, the temperature is controlled to be 25.5-26.5 ℃, and the pH is controlled to be 4-4.5.

The obtained THA-1 and THA-2 strains are respectively cultured according to the induction expression method, the obtained fermentation supernatant is detected, and the detection results are compared.

Electrophoretic detection of proteins

The expression of THA in the fermentation supernatant of the THA-1 strain and the double-site insert THA-2 strain was analyzed by Tricine-SDS-PAGE protein electrophoresis, and the results of the protein electrophoresis are shown in FIG. 4, wherein the symbol "M" represents Marker, the symbols "1", "2" and "3" represent the fermentation supernatants of the THA-1 strain after 48, 72 and 96 hours of methanol induction, respectively, and the symbols "4", "5" and "6" represent the fermentation supernatants of the THA-2 strain after 48, 72 and 96 hours of methanol induction, respectively. As can be seen from the figure, the protein bands of the fermentation supernatants of the THA-1 and THA-2 strains are both between 2000 and 4000Da, and the molecular weight of the protein bands is similar to the theoretical molecular weight of THA. In addition, it can be seen that the THA expression level of the THA-2 strain is significantly higher than that of the THA-1 strain.

Reversed phase high performance liquid chromatography detection

Detecting with Agilent 1260 type high performance liquid chromatograph, and loading sample to C18 reversed phase chromatographic column (Agilent ZORBAX SB-C18250 × 4.6mm, particle diameter 5 um). Elution was performed with a gradient of 0% to 80% acetonitrile containing 0.1% TFA for 30 minutes at a flow rate of 1ml/min, and the UV absorption peak at 220nm was recorded. The standard was 0.5mg/ml THA, and the loading was 50 ul. The THA fermentation supernatant was subjected to high speed centrifugation and filtration through a 0.45um pore size membrane, and the amount of the supernatant was 25 ul. The chromatogram peak is shown in FIG. 5, wherein FIG. 5A is the chromatogram result of 50ul THA standard substance with concentration of 0.5mg/mL, FIG. 5B is the chromatogram result of 25ul THA-1 strain fermentation supernatant, FIG. 5C is the chromatogram result of 25ul THA-2 strain fermentation supernatant, the THA content in the fermentation liquid is calculated according to peak area integral, the THA concentration in the fermentation supernatant fermentation liquid after THA-1 strain fermentation induction for 96 hours is 0.76mg/mL, and the THA concentration in the fermentation supernatant after THA-2 strain fermentation induction for 96 hours is 1.5 mg/mL.

Mass spectrometric detection

In order to accurately determine the molecular weight of THA polypeptide expressed by fermentation, THA (concentration of 1mg/mL) separated and purified by reversed-phase HPLC is sent to Beijing Minda micro-structured analysis and test center, Shimadzu high-resolution MALDI-TOF mass spectrometer is used, alpha-cyano-4-hydroxycinnamic acid (CHCA) is used as a matrix, the monoisotopic mass of the purified THA is determined under a positive ion reflection mode, the mass spectrum result is shown in FIG. 6, and the accurate molecular weight is determined to be 2434.75Da and is consistent with the theoretical molecular weight 2434.29 calculated according to the molecular formula.

Detection of bacteriostatic activity of fermentation supernatant

The antibacterial activity of the fermentation supernatant after THA-1 and THA-2 strains are respectively induced for 96 hours to escherichia coli ATCC25922 is detected, fig. 7 is an antibacterial activity test result, the labels "1" and "2" in the figure respectively represent the fermentation supernatant before the THA-1 and THA-2 strains are induced, the labels "3" and "4" respectively represent the fermentation supernatant after the THA-1 and THA-2 strains are induced for 96 hours, and the sample amount of each hole in the figure is 10 ul. As can be seen from the figure, the fermentation supernatants of THA-1 and THA-2 strains after 96 hours of induction both had significant bacteriostatic activity.

Evaluation of Strain stability

The strain stability was evaluated by serial subculture. The evaluation method is as follows: the strain was inoculated into 3mL of BMGY medium and cultured at 30 ℃ and 220rpm for 24 hours to prepare a seed solution. 0.5mL of the seed solution was inoculated into 25mL of BMGY medium and cultured at 30 ℃ and 220rpm for 24 hours. The bacterial solution was transferred to a 50mL centrifuge tube, centrifuged at 6000rpm for 5min, the supernatant was discarded, then the cells were suspended in 25mL BMMY medium with 1% methanol concentration, the bacterial solution was transferred to a 250mL Erlenmeyer flask, cultured at 30 ℃ and 220rpm for 72 hours, and methanol was added to 1% concentration every 24 hours. And after the induction is finished, taking the fermentation liquor, centrifuging at 8000rpm for 5min, and taking the supernatant to perform the Minimum Inhibitory Concentration (MIC) detection of THA.

The THA-2 strain is subjected to continuous subculture for 10 generations, the minimum inhibitory concentration of the fermentation supernatant is detected, and the result shows that the MIC of the shake flask fermentation supernatant of each generation of strain is 1/80, which indicates that the strain stability is good.

By combining the detection results, the recombinant Pichia pastoris (Pichia pastoris) THA-2 strain constructed by the method of multi-site insertion can stably express and secrete THA with bacteriostatic activity, the THA expression level of the strain induced for 96 hours can reach 1.5g/L, which is more than 2 times that of the prior art, and the strain has good genetic stability and high application value.

The recombinant Pichia pastoris (Pichia pastoris) THA-2 strain is preserved in Guangdong province microbial strain preservation center 10, 12 months in 2021, and the preservation number is GDMCC No.61977, at the institute of microorganisms in Guangdong province, No. 59 floor, No. 5 floor, No. 59 of Chongli Zhou province, No. 100 of Guangzhou city.

It should be understood that equivalents and modifications of the technical solution and inventive concept thereof may occur to those skilled in the art, and all such modifications and alterations should fall within the scope of the appended claims.

Sequence listing

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Claims (10)

1. The recombinant Pichia pastoris for secretory expression of recombinant stink bug defensin THA is characterized in that the preservation number of the recombinant Pichia pastoris (Pichia pastoris) is GDMCC No. 61977.

2. A method for constructing recombinant Pichia pastoris, which is used for constructing the recombinant Pichia pastoris for secretory expression of recombinant stinkbug defensin THA according to claim 1, and comprises the following steps:

step S001, designing a recombinant stink bug defensin THA coding gene: using a THA mature peptide sequence as a target product, and designing a THA coding gene according to pichia pastoris codon preference;

step S002, constructing an expression vector: respectively connecting the THA coding gene designed in the step S001 to vectors pPICZ alpha A and pPIC3.5K, and respectively constructing expression vectors pPICZ alpha A-THA and pPIC3.5K-THA;

step S003, constructing recombinant pichia pastoris: respectively carrying out linearization treatment on the pPICZ alpha A-THA and the pPIC3.5K-THA expression vectors, then transferring the pPICZ alpha A-THA linearization expression vectors into pichia pastoris competent cells, inoculating the converted pichia pastoris into a screening culture medium for culture, detecting the strain expression quantity after obtaining single strains, and selecting an optimal strain THA-1; and then transferring the pPIC3.5K-THA linear expression vector into a strain THA-1 competent cell, performing strain expression amount detection again after obtaining single strains through screening culture medium culture, and selecting an optimal strain THA-2 to obtain the recombinant pichia pastoris for secreting and expressing the recombinant stinkbug defensin THA.

3. The method for constructing recombinant pichia pastoris according to claim 2, wherein in step S001, the sequence of the gene encoding THA is SEQ ID No. 2.

4. The method for constructing recombinant pichia pastoris according to claim 2, wherein in the step S002, the construction of the pPICZ α a-THA expression vector comprises the following steps: adding an XhoI sequence at the 5 'end of the THA coding gene sequence, adding an XbaI sequence at the 3' end, performing double digestion treatment by using restriction enzymes XhoI and XbaI, and connecting the THA coding gene subjected to double digestion treatment to a linearized pPICZ alpha A vector subjected to double digestion treatment by using the XhoI and XbaI by using T4DNA ligase to construct the pPICZ alpha A-THA expression vector.

5. The method for constructing recombinant pichia pastoris according to claim 2, wherein in the step S002, the construction of the ppic3.5k-THA expression vector comprises the following steps: adding an alpha-factor secretion signal peptide sequence at the 5 'end of a THA coding gene sequence, then adding a BamHI sequence in front of an initiation codon of the signal peptide, adding a NotI sequence at the 3' end of the THA coding gene sequence to obtain a fusion sequence, then carrying out double digestion treatment by using restriction enzymes BamHI and NotI, and then connecting the THA coding gene subjected to double digestion treatment to a linearized pPIC3.5K vector which is also subjected to double digestion treatment by using BamHI and NotI by using T4DNA ligase to construct the pPIC3.5K-THA expression vector.

6. The method for constructing recombinant pichia pastoris according to claim 2, wherein in step S003, the pPICZ α a-THA and ppic3.5k-THA linearized expression vectors are transferred into competent cells by electroporation.

7. The method for constructing recombinant pichia pastoris according to claim 2, wherein in step S003, the pPICZ α a-THA expression vector is linearized with a PmeI restriction enzyme.

8. The method for constructing recombinant pichia pastoris according to claim 2, wherein in the step S003, the ppic3.5k-THA expression vector is linearized with a SalI restriction enzyme.

9. The method for constructing recombinant pichia pastoris according to claim 2, wherein in the step S003, the strain expression level detection comprises the following steps: and (3) selecting a single colony for culturing, inducing the single colony for 48 hours by using 1% methanol after the single colony is stabilized, collecting fermentation supernatant, centrifuging the supernatant, detecting the size of a bacteriostatic zone, and selecting a strain with the optimal expression quantity.

10. An inducible expression method of recombinant pichia pastoris, which is used for the recombinant pichia pastoris of claim 1 to secrete and express the recombinant stinkbug defensin THA, and comprises the following steps:

step S101, primary culture: inoculating the recombinant pichia pastoris into 25mL of BMGY medium, and culturing for 24 hours at 30 ℃ and 220rpm to obtain a first-stage seed solution;

step S102, secondary culture: inoculating 20mL of the first-stage seed solution into 200mL of BMGY culture medium, and culturing at 30 ℃ and 220rpm for 24 hours to obtain a second-stage seed solution;

step S103, three-stage culture: inoculating the second-stage seed liquid into a fermentation tank containing 2L BSM culture medium, and culturing at 29.5-30.5 deg.C, 19.5-20.5% dissolved oxygen and 4.5-5.5% pH;

step S104, induced expression: when the glycerol in the BSM culture medium is exhausted, 10% of glycerol is fed, after the glycerol is exhausted, the dissolved oxygen rises to 100%, starving is carried out for 1 hour, then methanol is fed to induce THA expression, the induction time is 96 hours, the temperature is controlled to be 25.5-26.5 ℃, and the pH is controlled to be 4-4.5.

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