KR20090038260A - A method of intestinal delivery of therapeutic proteins using conditional suicide of genetically modified microorganisms - Google Patents

Abstract

A method for delivering therapeutic proteins using conditional suicide of genetically modified microorganisms to small intestine is provided to safely deliver therapeutic proteins to small intestine by using Pichia pastoris JW501 strain introduced with a catalytic domain gene of diphtheria toxin. A method for preparing Pichia pastoris transformant is to induce conditional suicide under the control of an AOX1 promoter by introducing a gene coding a catalytic domain 1-185 amino acid residue of diphtheria toxin to a wild type Pichia pastoris strain. The transformant additionally comprises an encoding gene of fusion protein consisting of the Fc domain of human growth hormone and human immunoglobulin 1, GAP promoter and alpha mating prepro secretional signal peptide. The conditional suicide occurs in the control condition of AOX1(alcohol oxidase 1) promoter which uses ethanol or glucose as a carbon source and is induced with formic acid of 0.075%.

Description

A method of intestinal delivery of therapeutic proteins using conditional suicide of genetically modified microorganisms}

The present invention relates to a method for intestinal delivery of therapeutic proteins using conditional suicide of genetically modified microorganisms. More specifically, the present invention relates to a method for safely secreting therapeutic proteins in the small intestine by conditionally suicide under specific induction conditions using yeast recombined with catalytic domain genes of diphtheria toxin and therapeutic proteins.

Genetically modified microorganisms (GMMs) can be used in a variety of applications, including the production of drugs, vitamins and enzymes, bioremediation and agricultural use. In particular, genetically modified food-grade microorganisms can be used to deliver therapeutic proteins to the mucosa for disease treatment and immune induction. However, when GMMs release to the environment, they can survive and proliferate, and can also modify other microorganisms by delivering antibiotic resistance genes or other genes. Auxotrophic strains or strains that conditionally express toxin proteins have been used to inhibit the spread of GMMs. Among these strategies, Timmy delay agent synthase gene (thymidylate synthase gene), the thyA deficient Lactococcus lactis (Lactococcus lactis ) effectively induced the death of GMM upon thymidine or thymine depletion. Further, thymine or thymidine dependence Lactococcus lactis (Lactococcus expressing human IL-10 lactis ) Thy12 strain is in clinical trials for the treatment of Crohn's disease.

Although thymidine or thymine-dependent recombinant Lactococcus lactis bacteria have an attractive and effective containment system, the secretion rate of heterologous proteins is low and the technique can be applied to other therapeutic proteins of large molecular weight. It is difficult to apply to small intestine delivery.

In order to overcome the above limitations of the prior art, the present invention aims to provide Pichia pastoris capable of conditionally suicide.

Another object of the present invention is to provide a method for delivering a therapeutic protein to the small intestine using the transformant.

The object of the present invention is to introduce a gene of a therapeutic protein and a catalytic domain gene of diphtheria toxin to construct Pichia pastoris which conditionally commits suicide under the control of the induction promoter AOX1, and the growth profile of the transformant This was achieved by investigating the file and genetic stability, investigating the optimal conditions for induction of the inducible promoter AOX1, and examining whether the transformant secreted the therapeutic protein.

A feature of the present invention is to provide a method for the safe delivery of therapeutic proteins to the small intestine through the suicide of microorganisms by conditional induction under the control of the inducible promoter AOX1 (alcohol oxidase 1), and to provide a method for inhibiting the survival and proliferation of genetically modified microorganisms. It is done.

The microorganism used for the conditional suicide of the microorganism of the present invention is Pichia pastoris , and the catalytic domain of diphtheria toxin was used for the conditional suicide. Since one active molecule is sufficient to kill one cell, it can kill the cell through intracellular expression of the catalytic domain by conditional induction. In addition, since the catalytic domain of diphtheria toxin inactivates protein synthesis in eukaryotic cells by ADP-ribosylation of Elongation Factor-2 (EF-2), Expression will immediately block protein synthesis and consequently accumulate only trace amounts of catalytic domains within the cell. Also, because it does not have a secretory signal sequence, no accidental escape of the catalytic domain from the cytosol to the culture medium or the small intestine of humans and livestock occurs. Thus, the escaped catalytic domain is not harmful to humans and livestock because the catalytic domain cannot bind to the cell and cannot migrate to the cytosol.

We chose Pichia pastoris as a tool for delivery of therapeutic proteins to the small intestine for the following reasons:

a) free of endotoxin and retrovirus,

b) effective protein secretion,

c) can be cultured at high cell density (30-40%),

d) Saccharomyces cerevisiae ), generally recognized as safe (GRAS),

e) has a strong inducible promoter (alcohol oxidase 1),

f) relatively resistant to diphtheria toxin.

Because of their resistance to diphtheria toxins, the inventors have succeeded in developing Pichia pastoris that can conditionally commit suicide.

Conditionally suicide of the present invention Pichia Pastoris pastoris ) transformants secrete coding genes, GAP promoters and alpha mating prepros, of genes encoding catalytic domains 1-185 amino acid residues of diphtheria toxin, fusion proteins consisting of human growth hormone and Fc regions of human immunoglobulin 1 It is characterized in that it comprises a signal peptide (alpha mating prepro secretional signal peptide).

In the present invention, the conditional suicide of the transformant was implemented under the control of the inducible promoter AOX1 (alcohol oxidase 1).

The new strain, JW501, survived only with the presence of yeast extract with or without methanol, the inducer. Therefore, it can be used as a tool for containment of genetically modified microorganisms because the JW501 strain will not be able to survive under the strict conditions of the septic tank for sewage treatment.

In addition, the secondary (formic acid) metabolite formic acid in methanol when to provide additional means for regulating the GMMs conditional to avoid suicide Chiapas Laboratories in the (Pichia pastoris ) was selectively killed at 0.075%. The selective killing effect of formic acid, a secondary metabolite of methanol, on the survival of this new strain, JW501, is thought to provide clues to inhibit the spread of genetically modified microorganisms.

Moreover, the new strain JW501 has the ability to secrete high molecular weight therapeutic proteins.

Thus, the present invention provides an effective method for blockade of GMMs and small intestinal delivery of therapeutic proteins.

Hereinafter, the present invention will be described in detail by way of examples, but the scope of the present invention is not limited only to these examples.

The present invention relates to a small intestine delivery method of a therapeutic protein using conditional suicide of a genetically modified microorganism, wherein a catalytic domain gene of diphtheria toxin, which can conditionally suicide under the control of an inducible promoter AOX1 and express a therapeutic protein, is introduced. There is an excellent effect of providing the strained Pichia pastoris JW501 strain. The present invention has an excellent effect of providing an effective method to safely deliver a therapeutic protein to the small intestine and to block genetically modified microorganisms by using the strain. Therefore, the present invention is a very useful invention in the pharmaceutical industry.

Hereinafter, the present invention will be described in detail with reference to the following examples, which are intended to explain the present invention in more detail, and the scope of the present invention is not limited to these examples.

Example  1: conditionally committing suicide Peachia Pastoris ( Pichia pastoris ) JW501 Manufacture

The catalytic domain of diphtheria toxin, which has an additional methionine residue on the N-terminus side, to prepare Pichia pastoris , in which the catalytic domain of diphtheria toxin is expressed in the cytosol and conditionally suicides (residue 1 ~ 185) gene was amplified by PCR to construct pPIC3 vector.

Escherichia as a Host for Genetic Engineering coli ) Nova Blue strain (Novazen, Madison, USA) was used and cultured in low salt LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl; 2% agar for plates; 50 μg / for plasmid selection) Ml ampicillin or 25 μg / ml zeosin).

The catalytic domain gene was amplified using DT390 (1 to 390 amino acid residues of diphtheria toxin), which is fully utilized as template DNA, and the primers are as follows:

Forward primer: 5'-atg ttcgaa acgATGGCTGGCGCTGATGATGTCGTC-3 '

Reverse primer: 5'-atg gaattc tcaGGCTTGAGCCATATACTCATA-3 '

To amplify the PCR product, denaturing at 94 ° C for 2 minutes, followed by 30 amplification cycles (30 seconds at 94 ° C, 30 seconds at 50 ° C, 1 minute at 72 ° C), and reacting at 72 ° C for 7 minutes The PCR reaction was terminated. Thereafter, PCR products were digested with Sfu I and EcoR I. The final DNA fragment was ligated to pnPICZα vector treated with Sfu I, EcoR I and cal intestinal phosphatase (CIP). From the gene construct, Small fragments digested with Sac I and EcoR I containing catalytic domain genes were subcloned into pPIC3 vectors (see cleavage map of FIG. 1 and SEQ ID NO: 1) treated with Sac I, EcoR I and CIP.

Next, a gene of the hGH-Fc fusion protein was constructed in the pGAPZα vector for expression of the hGH-Fc fusion protein. The protein consists of the human growth hormone and the Fc region of human IgG1 and is expressed in dimeric form outside the cell. First, hGH-h-Fc gene constructs for the expression of hGH-Fc fusion protein prepared in the previous study (Korean Patent No. 10-0594607) were cut with BstB I and EcoR I. the small fragment containing the hGH-Fc gene was cloned into the BstB I, pGAPZα vector treated with EcoR I and CIP (see Fig cleavage map and sequence 2 of 2) which is used to transform the strain JW501.

Next, 10 μg of the plasmid was made linear using Sac I, followed by electrophoresis using a Bio-Rad Gene Pulser Xcell electroporation system (Biorad Laboratories, Hercules, USA) at 200 W, 7,500 V / cm and 25 μF. Pichia Pastoris ( Pichia) pastoris) GS115 strains ^were inserted ((Mut ⁺ His, Invitrogen, USA Carlsbad) transformants are histidine-deficient RD agar plates (1M sorbitol, 2% dextrose woods, without 1.34% amino yeast nitrogen YPD agar plate (1% yeast extract, 2%) containing base, 4 × 10 ⁻⁵ % biotin, and 0.005% amino acid mixture; 2% agar for plate) or 100 μg / ml zeocin (Invitrogen) Peptone and 2% dextrose; 2% agar for plates).

Next, one colony for methanol-induced cultivation of Pichia pastoris in 250 ml (orbit diameter, 1.9 cm) at 28 ° C. in 5 ml YPD broth in a 14 ml snap-cap test tube. Incubate 3 ml of BMMY broth (1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH7.0, yeast nitrogen base without 1.34% amino acid, 4 × ^10-5 % biotin, 0.5% methanol and 1% casa) Resuspended in mino acid) and induced for 1 to 2 days by adding 15 μl of methanol every 24 hours.

For constitutive expression, one colony of Pichia pastoris was placed at 250 rpm (orbit diameter, 1.9 cm) at 28 ° C. in 5 ml YPD broth in a 14 ml snap-cap test tube for 1-2 days. Cultures and culture supernatants were analyzed.

Gene products were expressed in cells under the control of the methanol-induced AOX1 (alcohol oxidase 1) promoter. Even in the absence of methanol, the AOX1 promoter is known to induce expression at very slight levels, and because of the severe toxicity of the catalytic domain so expressed, only three transformants were obtained.

To confirm the conditional suicide, the transformants were subjected to a minimal medium MM agar plate (1.34% amino acid free yeast nitrogen base, 4 × 10 ⁻⁵ % biotin, 0.5% methanol and 2% agar) containing inducer methanol. Plate).

As expected, all transformants expressed a toxic catalytic domain and did not grow at all on MM agar plates. In addition, Western blotting revealed no bands of the catalytic domains in the supernatants of the cultures induced for 1 or 2 days (not shown). One of them was selected and named JW501. The transformant JW501 was deposited on October 1, 2007, at the KCTC in the Korea Research Institute of Bioscience and Biotechnology and was assigned accession number KCTC 11208BP.

Experimental Example  One: JW501  Growth profile and genetic stability of the strain

The growth profile of the new strain JW501 was examined in several agar plates containing methanol or glucose as the carbon source to determine which condition induced effective cell death. Pichia Pastoris pastoris ) X-33 strain (Mut ⁺ , Invitrogen, Carlsbad, USA) was used as a control.

Wet cell density (%, w / v) was measured to measure cell growth. To determine the growth profile of the yeast strain, MM agar plates (yeast nitrogen base without 1.34% amino acid, 4 × 10 ⁻⁵ % biotin, 0.5% methanol and 2% agar) or MD agar plates (yield without 1.34% amino acid) Base, 4 × 10 ⁻⁵ % biotin, 2% dextrose and 2% agar).

1 ml of the culture sample was injected into a previously weighed 1.5 ml-microcentrifuge tube, and centrifuged at 20,800 × g and 25 ° C. for 2 minutes. The supernatant was removed by pipette and the remaining liquid in the tube was removed by filter paper. After weighing the tube containing the cell pellet, the cell density (%, w / v) was calculated.

For spot analysis, test strains were incubated for 30 hours at 28 ° C., 250 rpm in 5 ml of YPD medium to compare growth profiles on agar plates containing various media components. The absorbance of each culture was measured at 600 nm. The culture was adjusted to phosphate buffered saline (PBS), pH 7.4 to 2.0 at OD ₆₀₀ . Five dilutions were serially diluted (so that the OD ₆₀₀ was 2.0, 0.4, 0.08, 0.016, 0.032, and 0.00064) and 4 μl of diluted culture was spotted onto various agar plates. The plates were incubated at 28 ° C. for 2-4 days until spot colonies appeared and the results are shown in FIG. 3. In FIG. 3, MM means minimum methanol medium, MD means minimum dextrose medium, and YMM means MM + 1% yeast extract.

In spot analysis, minimal medium agar plates containing methanol or glucose induced complete killing of JW501 (FIG. 3). However, the minimal medium containing 1% yeast extract (w / v) and methanol failed to induce the death of JW501, suggesting that the yeast extract has some protective capacity against diphtheria toxin.

In the case of JW501, a novel strain of the invention, it is expressed in cells under the control of the AOX1 promoter, which is known to be tightly regulated by methanol.

In the presence of glycerol, mRNA of AOX1 by the AOX1 promoter is not detected in the northern blot. However, in the absence of a carbon source, low levels of AOX1 mRNA are detected in the northern blot. Thus, we predicted that the presence of glycerol and / or glucose would block the toxicity of the catalytic domain of diphtheria toxin, but glycerol and glucose did not affect the growth of the JW501 strain. This may be related to the nature of the catalytic domain that one active substance is sufficient to kill cells. Although in the presence of glycerol the mRNA of AOX1 was not detected in the Northern blot, even an undetectable amount of mRNA is sufficient to synthesize one active catalytic domain. Thus, the JW501 strain may not grow in glucose agar plates.

Instead, yeast extract showed protective activity against catalytic domain in Pichia pastoris . In a previous study, the inventors observed that continuous application of yeast extract attenuated the toxicity of immune toxins during methanol induction to produce anti-T cell immune toxins in wild-type Pichia pastoris . have. Diphtheria toxin consists of three domains: catalytic domain, translocation domain and recognition domain. To kill eukaryotic cells, the cognitive domain of diphtheria toxin is first bound to a heparin binding EGF-like receptor on the cell surface. This binding induces endocytosis by the receptor followed by rapid localization to the endosomes. The translocation domain of the toxin changes shape in the acidic environment of the endosome and inserts it into the endosome membrane. The translocation domain interacts with Hsp90 and thioredoxin reductase of the cytosol to relocate the catalytic domain to the cytosol. The migrated catalytic domains refold and enzymatically inactivate the synthesis of intracellular proteins following ADP-ribosylation of EF-2. In this intoxication mechanism, several inhibitors have been reported. Ammonium chloride and monensin inhibit certain steps in the site shift process to inhibit acidification of endosomes or chloroquines. As a result, the position shift from the endosome of the catalytic domain to the cytosol fails. However, no inhibitory activity of the catalytic domain has been reported. Thus, the inventors could infer that since the yeast extract has numerous low molecular peptides, one or several peptides will inhibit the enzymatic activity of the catalytic domain.

On the other hand, since bile salts often delay the growth of lactic acid bacteria for probiotic use, it was tested whether bile salts delayed the growth of JW501 strains. Oxgall extract was used as a source of bile salts. JW501 cultures incubated for 30 hours were inoculated three times in 5 ml of YPD medium containing 0-1.0% oxalic extract. Cell density was measured by time zone and the results are shown in FIG. 4. Error bars represent SD (standard deviation).

As shown in Figure 4, oxgal extract did not affect the growth of JW501 strain in the range of 0 ~ 1.0% (w / v).

Passage was cultured on YPD agar plates to investigate the genetic stability of the JW501 strain. To passage the JW501 strain, initial colonies on the selection agar plates for transformant screening were designated as passage number 1. This colony was streaked on fresh YPD agar plates and incubated at 28 ° C. for 2 days. Well separated colonies (passage 2) were randomly picked and streaked on other fresh YPD agar plates and incubated. This passage was repeated 60 times until a JW501 colony with passage number 60 was obtained. One colony from each YPD plate for subculture was examined for growth profiles in minimal medium agar plates containing glucose.

All colonies tested did not grow on minimal medium agar plates. This suggests that the JW501 strain is genetically stable even if it contains the virulence gene of the catalytic domain of diphtheria toxin.

Experimental Example  2: AOX1  Promoter condition of promoter

Since formic acid and hydrogen peroxide are secondary metabolites in the methanol metabolism of Pichia pastoris , we investigated whether formic acid or hydrogen peroxide induces the AOX1 promoter.

To this end, the blood hGH-Fc fusion protein expressing Chiapas pastoris (Pichia pastoris) transformants (H6 strain, Lee, CH et al. 2007. Expression and characterization of human growth hormone-Fc fusion proteins for transyctosis induction. Biotechnol ... Appl Biochem 46 (4 ): 211-217 was used). H6 strains were used to compare expression levels in various induction media including formic acid, formic acid and dextrose, hydrogen peroxide, or hydrogen peroxide and dextrose. Since methanol is known as an inducer for the AOX1 promoter, and dextrose inhibits the AOX1 promoter, methanol and dextrose were used as positive and negative controls, respectively.

Next, the proteins were separated on 4-20% Tris-glycine precast SDS-PAGE gel (Invitrogen) under non-reducing or reducing conditions and transferred to nitrocellulose membranes by electroblotting. Nonspecific binding was blocked with 5% non-fat skimmed milk dissolved in Tris-buffered saline buffer (50 mM Tris-HCl, pH7.5, 150 mM NaCl) containing 0.1% Tween-20.

To detect recombinant hGH-Fc fusion protein, peroxidase-labeled goat anti-human immunoglobulin (Fc) antibody (1: 1000 dilution; custom antibody of Biosynthesis Incorporated) and peroxidase- Labeled goat anti-rabbit immunoglobulin (H + L) antibodies (1: 5000 dilution; KPL) were used as primary and secondary antibodies, respectively. Bands of the catalytic domain of the hGH-Fc fusion protein were observed with one-step NBT / BCIP substrates (Pierce Chemical Company, Roxford, USA). The results are shown in FIG. FIG. 5A shows Coomassie-stained SDS-PAGE, M12 shows Mark12 wide-range protein markers (Invitrogen), YP for 1% yeast extract and 2% peptone, YPM for YP + 0.5% methanol, YPD for YP + 2% dextrose, YPF stands for YP + 0.5% formic acid, YPDF stands for YPD + 0.5% formic acid, YPH stands for YP + 0.5% hydrogen peroxide, and YPDH stands for YPD + 0.5% hydrogen peroxide. Figure 5B shows the Western blotting results of hydrogen peroxide induced culture, HP means hydrogen peroxide, SeeB means SeeBlue Plus2 prestained protein makers (Invitrogen). Arrows indicate bands of dimeric hGH-Fc fusion proteins.

As shown in FIG. 5A, the hGH-Fc fusion protein was only expressed at detectable levels on Coomassie stained gels.

Next, the concentration of hydrogen peroxide that can induce the expression of the hGH-Fc fusion protein was investigated.

As shown in FIG. 5B, hydrogen peroxide at a concentration of 0.05-0.5% induced the AOX1 promoter. Even in the absence of hydrogen peroxide, the AOX1 promoter was induced.

Next, the effects of formic acid and hydrogen peroxide on the growth of the JW501 strain were evaluated by concentration. Spot analysis was used to compare growth profiles of JW501 strains and X-33 strains on YP plates containing 0.075% formic acid or 0.075% hydrogen peroxide.

As shown in FIG. 6A, formic acid killed the JW501 strain at a concentration of 0.075%, but did not kill for the X-33 host strain.

However, at the same concentration, hydrogen peroxide killed both JW501 and X-33 strains (FIG. 6B).

Experimental Example  3: JW501  In strain hGH - Fc  Expression of Fusion Proteins

The ability of the JW501 strain to secrete hGH-Fc fusion protein was evaluated. To this end, a gene encoding the hGH-Fc fusion protein was constructed in the pGAPZα vector. Recombinant genes were expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter and secretion of the gene product hGH-Fc fusion protein was directed by an alpha mating prepro secretional signal peptide. It was. The result is shown in FIG. FIG. 7A shows Coomassie-stained SDS-PAGE results and FIG. 7B shows Western blotting results. Each number represents the transformant identification number and the arrow represents the band of the dimeric hGH-Fc fusion protein. M12 stands for Mark12 wide-range protein makers (Invitrogen) and SeeB stands for SeeBlue Plus2 prestained protein makers (Invitrogen).

As shown in Figure 7, it was confirmed that the JW501 strain can express a heterologous protein.

1 is a cleavage map of a pPIC3 vector.

2 is a cleavage map of the pGAPZα vector.

Figure 3 shows a comparison of the growth profile of JW501 and X-33 strain in various growth media using the spot analysis.

Figure 4 shows the effect of oxgal extract on the growth of the JW501 strain.

Figure 5 shows the expression of hGH-Fc fusion protein obtained in H6 strain in various induction medium, A is Coomassie-stained SDS-PAGE, B is Western blotting results of hydrogen peroxide induction culture.

Figure 6 shows the selective killing effect of formic acid on the JW501 strain.

Figure 7 shows the constitutive expression of hGH-Fc fusion protein in JW501 strain, A shows Coomassie-stained SDS-PAGE results, B shows Western blotting results.

<110> INSILICOTECH CO. LTD. <120> A method of intestinal delivery of therapeutic proteins using          conditional suicide of genetically modified microorganisms <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 8306 <212> DNA <213> Artificial Sequence <220> <223> vector <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 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cagccagatc 2340 catcactgct tggccaatat gtttcagtcc ctcaggagtt acgtcttgtg aagtgatgaa 2400 cttctggaag gttgcagtgt taactccgct gtattgacgg gcatatccgt acgttggcaa 2460 agtgtggttg gtaccggagg agtaatctcc acaactctct ggagagtagg caccaacaaa 2520 cacagatcca gcgtgttgta cttgatcaac ataagaagaa gcattctcga tttgcaggat 2580 caagtgttca ggagcgtact gattggacat ttccaaagcc tgctcgtagg ttgcaaccga 2640 tagggttgta gagtgtgcaa tacacttgcg tacaatttca acccttggca actgcacagc 2700 ttggttgtga acagcatctt caattctggc aagctccttg tctgtcatat cgacagccaa 2760 cagaatcacc tgggaatcaa taccatgttc agcttgagac agaaggtctg aggcaacgaa 2820 atctggatca gcgtatttat cagcaataac tagaacttca gaaggcccag caggcatgtc 2880 aatactacac agggctgatg tgtcattttg aaccatcatc ttggcagcag taacgaactg 2940 gtttcctgga ccaaatattt tgtcacactt aggaacagtt tctgttccgt aagccatagc 3000 agctactgcc tgggcgcctc ctgctagcac gatacactta gcaccaacct tgtgggcaac 3060 gtagatgact tctggggtaa gggtaccatc cttcttaggt ggagatgcaa aaacaatttc 3120 tttgcaacca gcaactttgg caggaacacc cagcatcagg gaagtggaag gcagaattgc 3180 ggttccacca ggaatataga ggccaacttt ctcaataggt cttgcaaaac gagagcagac 3240 tacaccaggg caagtctcaa cttgcaacgt ctccgttagt tgagcttcat ggaatttcct 3300 gacgttatct atagagagat caatggctct cttaacgtta tctggcaatt gcataagttc 3360 ctctgggaaa ggagcttcta acacaggtgt cttcaaagcg actccatcaa acttggcagt 3420 tagttctaaa agggctttgt caccattttg acgaacattg tcgacaattg gtttgactaa 3480 ttccataatc tgttccgttt tctggatagg acgacgaagg gcatcttcaa tttcttgtga 3540 ggaggcctta gaaacgtcaa ttttgcacaa ttcaatacga ccttcagaag ggacttcttt 3600 aggtttggat tcttctttag gttgttcctt ggtgtatcct ggcttggcat ctcctttcct 3660 tctagtgacc tttagggact tcatatccag gtttctctcc acctcgtcca acgtcacacc 3720 gtacttggca catctaacta atgcaaaata aaataagtca gcacattccc aggctatatc 3780 ttccttggat ttagcttctg caagttcatc agcttcctcc ctaattttag cgttcaacaa 3840 aacttcgtcg tcaaataacc gtttggtata agaaccttct ggagcattgc tcttacgatc 3900 ccacaaggtg gcttccatgg ctctaagacc ctttgattgg ccaaaacagg aagtgcgttc 3960 caagtgacag aaaccaacac ctgtttgttc aaccacaaat ttcaagcagt ctccatcaca 4020 atccaattcg atacccagca acttttgagt tgctccagat gtagcacctt tataccacaa 4080 accgtgacga cgagattggt agactccagt ttgtgtcctt atagcctccg gaatagactt 4140 tttggacgag tacaccaggc ccaacgagta attagaagag tcagccacca aagtagtgaa 4200 tagaccatcg gggcggtcag tagtcaaaga cgccaacaaa atttcactga cagggaactt 4260 tttgacatct tcagaaagtt cgtattcagt agtcaattgc cgagcatcaa taatggggat 4320 tataccagaa gcaacagtgg aagtcacatc taccaacttt gcggtctcag aaaaagcata 4380 aacagttcta ctaccgccat tagtgaaact tttcaaatcg cccagtggag aagaaaaagg 4440 cacagcgata ctagcattag cgggcaagga tgcaacttta tcaaccaggg tcctatagat 4500 aaccctagcg cctgggatca tcctttggac aactctttct gccaaatcta ggtccaaaat 4560 cacttcattg ataccattat tgtacaactt gagcaagttg tcgatcagct cctcaaattg 4620 gtcctctgta acggatgact caacttgcac attaacttga agctcagtcg attgagtgaa 4680 cttgatcagg ttgtgcagct ggtcagcagc atagggaaac acggcttttc ctaccaaact 4740 caaggaatta tcaaactctg caacacttgc gtatgcaggt agcaagggaa atgtcatact 4800 tgaagtcgga cagtgagtgt agtcttgaga aattctgaag ccgtattttt attatcagtg 4860 agtcagtcat caggagatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac 4920 aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca 4980 cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg 5040 actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct 5100 caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgagtatc 5160 tatgattgga agtatgggaa tggtgatacc cgcattcttc agtgtcttga ggtctcctat 5220 cagattatgc ccaactaaag caaccggagg aggagatttc atggtaaatt tctctgactt 5280 ttggtcatca gtagactcga actgtgagac tatctcggtt atgacagcag aaatgtcctt 5340 cttggagaca gtaaatgaag tcccaccaat aaagaaatcc ttgttatcag gaacaaactt 5400 cttgtttcga actttttcgg tgccttgaac tataaaatgt agagtggata tgtcgggtag 5460 gaatggagcg ggcaaatgct taccttctgg accttcaaga ggtatgtagg gtttgtagat 5520 actgatgcca acttcagtga caacgttgct atttcgttca aaccattccg aatccagaga 5580 aatcaaagtt gtttgtctac tattgatcca agccagtgcg gtcttgaaac tgacaatagt 5640 gtgctcgtgt tttgaggtca tctttgtatg aataaatcta gtctttgatc taaataatct 5700 tgacgagcca aggcgataaa tacccaaatc taaaactctt ttaaaacgtt aaaaggacaa 5760 gtatgtctgc ctgtattaaa ccccaaatca gctcgtagtc tgatcctcat caacttgagg 5820 ggcactatct tgttttagag aaatttgcgg agatgcgata tcgagaaaaa ggtacgctga 5880 ttttaaacgt gaaatttatc tcaagatctc tgcctcgcgc gtttcggtga tgacggtgaa 5940 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg 6000 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg 6060 acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga 6120 ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat 6180 accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc 6240 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg 6300 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 6360 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 6420 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 6480 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 6540 ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg 6600 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 6660 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 6720 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 6780 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc 6840 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 6900 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 6960 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 7020 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt 7080 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc 7140 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 7200 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg 7260 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc 7320 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta 7380 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg 7440 ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 7500 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta 7560 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg 7620 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga 7680 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt 7740 gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca 7800 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt 7860 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt 7920 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 7980 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt 8040 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 8100 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa 8160 cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattaatt ctcatgtttg 8220 acagcttatc atcgataagc tgactcatgt tggtattgtg aaatagacgc agatcgggaa 8280 cactgaaaaa taacagttat tattcg 8306 <210> 2 <211> 4443 <212> DNA <213> Artificial Sequence <220> <223> vector <400> 2 agatcttttt tgtagaaatg tcttggtgtc ctcgtccaat caggtagcca tctctgaaat 60 atctggctcc gttgcaactc cgaacgacct gctggcaacg taaaattctc cggggtaaaa 120 cttaaatgtg gagtaatgga accagaaacg tctcttccct tctctctcct tccaccgccc 180 gttaccgtcc ctaggaaatt ttactctgct ggagagcttc ttctacggcc cccttgcagc 240 aatgctcttc ccagcattac gttgcgggta aaacggaggt cgtgtacccg acctagcagc 300 ccagggatgg aaaagtcccg gccgtcgctg gcaataatag cgggcggacg catgtcatga 360 gattattgga aaccaccaga atcgaatata aaaggcgaac acctttccca attttggttt 420 ctcctgaccc aaagacttta aatttaattt atttgtccct atttcaatca attgaacaac 480 tatttcgaaa cgatgagatt tccttcaatt tttactgctg ttttattcgc agcatcctcc 540 gcattagctg ctccagtcaa cactacaaca gaagatgaaa cggcacaaat tccggctgaa 600 gctgtcatcg gttactcaga tttagaaggg gatttcgatg ttgctgtttt gccattttcc 660 aacagcacaa ataacgggtt attgtttata aatactacta ttgccagcat tgctgctaaa 720 gaagaagggg tatctctcga gaaaagagcc ttcccaacca ttcccttatc cagattgttc 780 gacaacgcta tgttgagagc ccacagactg caccagctgg cctttgacac ctaccaggag 840 tttgaagaag cctatatccc aaaggaacag aagtattcat tcctgcagaa cccccagacc 900 tccctctgtt tctcagagtc tattccgaca ccctccaaca gggaggaaac acaacagaaa 960 tccaacctag agctgctccg catctccctg ctgctcatcc agtcgtggct ggagcccgtg 1020 cagttcctca ggagtgtctt cgccaacagc ctggtgtacg gcgcctctga cagcaacgtc 1080 tatgacctcc taaaggacct agaggaaggc atccaaacgc tgatggggag gctggaagat 1140 ggcagccccc ggactgggca gatcttcaag cagacctaca gcaagttcga cacaaactca 1200 cacaacgatg acgcactact caagaactac gggctgctct actgcttcag gaaggacatg 1260 gacaaggtcg agacattcct gcgcatcgtg cagtgccgct ctgtggaggg cagctgtggc 1320 ttcggtggag gtggatccga gcccaaatct tgtgacaaaa ctcacacatg cccaccgtgc 1380 ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac 1440 accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 1500 gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 1560 aagccgcggg aggagcagta caacagcacg taccgggtgg tcagcgtcct caccgtcctg 1620 caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 1680 gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 1740 accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc 1800 aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 1860 aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag 1920 ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 1980 gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaacaccac 2040 caccaccacc actgagaatt cacgtggccc agccggccgt ctcggatcgg tacctcgagc 2100 cgcggcggcc gccagctttc tagaacaaaa actcatctca gaagaggatc tgaatagcgc 2160 cgtcgaccat catcatcatc atcattgagt tttagcctta gacatgactg ttcctcagtt 2220 caagttgggc acttacgaga agaccggtct tgctagattc taatcaagag gatgtcagaa 2280 tgccatttgc ctgagagatg caggcttcat ttttgatact tttttatttg taacctatat 2340 agtataggat tttttttgtc attttgtttc ttctcgtacg agcttgctcc tgatcagcct 2400 atctcgcagc tgatgaatat cttgtggtag gggtttggga aaatcattcg agtttgatgt 2460 ttttcttggt atttcccact cctcttcaga gtacagaaga ttaagtgaga ccttcgtttg 2520 tgcggatccc ccacacacca tagcttcaaa atgtttctac tcctttttta ctcttccaga 2580 ttttctcgga ctccgcgcat cgccgtacca cttcaaaaca cccaagcaca gcatactaaa 2640 ttttccctct ttcttcctct agggtgtcgt taattacccg tactaaaggt ttggaaaaga 2700 aaaaagagac cgcctcgttt ctttttcttc gtcgaaaaag gcaataaaaa tttttatcac 2760 gtttcttttt cttgaaattt ttttttttag tttttttctc tttcagtgac ctccattgat 2820 atttaagtta ataaacggtc ttcaatttct caagtttcag tttcattttt cttgttctat 2880 tacaactttt tttacttctt gttcattaga aagaaagcat agcaatctaa tctaagggcg 2940 gtgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac aaggtgagga 3000 actaaaccat ggccaagttg accagtgccg ttccggtgct caccgcgcgc gacgtcgccg 3060 gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg gaggacgact 3120 tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg 3180 tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt 3240 ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg accgagatcg 3300 gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac tgcgtgcact 3360 tcgtggccga ggagcaggac tgacacgtcc gacggcggcc cacgggtccc aggcctcgga 3420 gatccgtccc ccttttcctt tgtcgatatc atgtaattag ttatgtcacg cttacattca 3480 cgccctcccc ccacatccgc tctaaccgaa aaggaaggag ttagacaacc tgaagtctag 3540 gtccctattt atttttttat agttatgtta gtattaagaa cgttatttat atttcaaatt 3600 tttctttttt ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt 3660 gagaaggttt tgggacgctc gaaggcttta atttgcaagc tggagaccaa catgtgagca 3720 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 3780 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 3840 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 3900 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 3960 tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 4020 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 4080 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 4140 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 4200 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 4260 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 4320 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 4380 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgcatgag 4440 atc 4443  

Claims (5)

The gene encoding the catalytic domain 1-185 amino acid residues of diphtheria toxin was expressed in wild type Pichia. pastoris ) Pichia under the conditional suicide under the control of the AOX1 promoter pastoris ) A method for producing a transformant. The method of claim 1, wherein the transformant is a coding gene, a GAP promoter and an alpha mating prepro secretional signal peptide of a fusion protein composed of human growth hormone and human immunoglobulin 1 Fc region. Method for producing a Pichia pastoris transformant, characterized in that it further comprises. The method of claim 1 or 2, wherein the conditional suicide of the transformant is characterized by the condition of regulation of the AOX1 (alcohol oxidase 1) promoter induced by 0.075% formic acid using methanol or glucose as a carbon source. Pichia Pastoris pastoris ) A method for producing a transformant. Pichia prepared according to the method of claims 1 or 2 pastoris ) JW501 strain. Pichia as described in claim 4 pastoris ) Small intestine delivery method of therapeutic protein using transformants.

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