CN104911203A - Industrial preparation method of recombinant human insulin - Google Patents
Industrial preparation method of recombinant human insulin Download PDFInfo
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- CN104911203A CN104911203A CN201510370509.XA CN201510370509A CN104911203A CN 104911203 A CN104911203 A CN 104911203A CN 201510370509 A CN201510370509 A CN 201510370509A CN 104911203 A CN104911203 A CN 104911203A
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Abstract
The invention discloses an industrial preparation method of recombinant human insulin, which comprises the following steps: S100: preparation of strain; S200: strain culture; S300: collection of inclusion body; S400: preliminary purification of RRhPI; S500: recombinant renaturation of RRhPI; S600: enzyme digestion transformation; S700: purification of hI; and S800: preparation of hI finished product. The method simplifies the technical process, enhances the production efficiency, improves the product quality, and is suitable for industrial preparation demands.
Description
Technical field
The present invention relates to biomedicine field, specifically refer to the industrial production process of a kind of recombinant human insulin.
Background technology
Along with the fast development of biotechnology, genetically engineered recombinant human insulin emerge, and comparatively animal-origin Regular Insulin advantage is more obvious, completely the same with the sequence of Regular Insulin in human body, and purity is higher, few containing host protein, safe without toxic side effect.
The preparation method of existing insulin human, complex procedures, to raw material and equipment requirements higher, the needs of mass production cannot be adapted to.
Summary of the invention
The object of the present invention is to provide the industrial production process of a kind of recombinant human insulin, simplification of flowsheet, enhance productivity, improve product quality, adapt to industrial preparation demand.
The present invention is achieved through the following technical solutions: the industrial production process of a kind of recombinant human insulin, comprises the following steps:
Step S100: prepare bacterial classification;
Step S200: cultivate bacterial classification;
Step S300: the collection of inclusion body; By bacterial classification freeze-drying below-20 DEG C of cultivating, then dissolve by buffer A, vibrate under room temperature state 2-4h, and collecting precipitation after solid-liquid separation, obtains inclusion body;
Step S400:RRhPI preliminary purification; After the inclusion body buffer B of collection is dissolved completely, again with buffer B and damping fluid C volume ratio for 1:2-1:10 adds damping fluid C, protein concentration is made to be 0.05-0.5mg/ml, then go up in buffer B balance DEAE-Sepharose FF post, use NaCl gradient elution, collect the elutriant containing RRhPI;
Step S500:RRhPI recombinates renaturation; RRhPI after preliminary purification takes off urea by Sephadex G-25, then is transformed in damping fluid D, places more than 18h, obtains RRhPI renaturation solution for 4 DEG C;
Step S600: enzyme cuts conversion; Be 2:1:2000 by the mass ratio of trypsinase, protaminase, albumen, in RRhPI renaturation solution, add trypsinase and protaminase, room temperature enzyme cuts 50-80min, then by 0.1mol/L ZnCl2 termination reaction and precipitation generates hI;
The purifying of step S700:hI; The purifying of hI comprises thick purifying and consummateization; During described thick purifying, by the two step column chromatographies of the hI in step S600 by the NaCl of Tris-HCl and 0-1.0mmol/L of preset 8-12mmol/L, preliminary purification is carried out to hI and obtains crude product; During described consummateization, carry out refiningly obtaining fine work to crude product with the chromatogram acetonitrile of 10-20mmol/L;
Step S800:hI finished product; Fine work enters filtration sterilization room, less than-20 DEG C freeze-drying again after repeatedly crystallization and washing, obtains hI finished product;
Described step S100 specifically refers to:
Step S110: with human pancreas's cDNA library for template, extracts goal gene by pcr amplification technology;
Step S120: by pQE-40 plasmid that alkaline lysis method of extracting ring fills from the intestinal bacteria cultivated by cell engineering;
Step S130: take out goal gene and pQE-40 plasmid respectively, utilize respectively restriction enzyme Bam HI and Hind III simultaneously enzyme cut and produce sticky end;
Step S140: below 20 DEG C, under the effect of T4DNA ligase enzyme, passes through sticky end by goal gene and pQE-40 Plasmids conjugation complementary, forms the recombinant DNA of an energy expression of insulin;
Step S150: the recombinant DNA containing goal gene is put into colibacillary nutrient solution and adds calcium chloride, makes intestinal bacteria be absorbed by recombinant DNA, recombinant DNA Transformed E .coli competent cell M15;
Described step S200 specifically refers to:
Step S210: take out the bacterial classification prepared in step S100 and activate;
Step S220: adopt second order fermentation mode, constant temperature oscillation 2-3h;
Step S230: induce RRhPI/pQE40 E.coli M15 bacterial classification to express RRhPI with the IPTG of 0.5mmol/L.
Further, described step S210 specifically refers to: getting RRhPI/pQE40 E.coli M15 slant strains one ring, to be inoculated in pH value be in the substratum of 6.8-7.6, and carry out shaking culture with the speed of 200-280rpm under room temperature condition, air flow is 1:1.3-1:1.6; The main component of described substratum is 3-8g/L Tryptones, 1-3g/L L-glutamic acid, 6-8g/L yeast extract, 0.1-1.0g/L ammonium sulfate, 2-3g/L glucose, 2-3g/L glycerine, 90-110mg/L penbritin, 40-60mg/L kantlex.
Further, the optimal proportion of described substratum is 5g/L Tryptones, 2g/L L-glutamic acid, 7g/L yeast extract, 0.5g/L ammonium sulfate, 2.5g/L glucose, 2.7g/L glycerine, 100mg/L penbritin, 50mg/L kantlex; Described substratum NaOH regulates pH value to 7.2.
Further, in described step S300, the main component of buffer A is 40-60mmol/L Tris-HCl, 0.2-0.8mmol/L EDTA, 40-60mmol/L NaCl, 5% glycerine, 0.1-0.5mmol/L DTT; The pH value of described buffer A is 7.8-8.2.
Further, in described step S300, the main component of buffer A is 50mmol/L Tris-HCl, 0.5mmol/L EDTA, 50mmol/L NaCl, 5% glycerine, 0.3mmol/L DTT; The pH value of described buffer A is 7.9.
Further, in described step S400, the main component of buffer B is 0.1%-0.3% β-thioglycol, 5-50mmol/L Tris-HCl, 5-10mol/L urea; The pH value of described buffer B is 7.8-8.2.
Further, in described step S400, the main component of buffer B is 0.2% β-thioglycol, 30mmol/L Tris-HCl, 8mol/L urea; The pH value of described buffer B is 8.0.
Further, in described step S400, the main component of damping fluid C is the halfcystine of 40-70mmol/L Gly-NaOH, 0.3%-0.5%; The pH value of described damping fluid C is 9-10.
Further, in described step S500, the main component of damping fluid D is 40-70mmol/L Gly-NaOH, 10-50mmol/L GSSG, 10-50mmol/L GSH; The pH value of described damping fluid D is 9-10.
Further, in described step S500, the main component of damping fluid D is 50mmol/L Gly-NaOH, 30mmol/L GSSG, 30mmol/L GSH; The pH value of described damping fluid D is 9.5.
The present invention compared with prior art, has the following advantages and beneficial effect: simplification of flowsheet, enhances productivity, and improves product quality, adapts to industrial preparation demand.
Accompanying drawing explanation
Fig. 1 is process flow diagram of the present invention.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1:
The industrial production process of a kind of recombinant human insulin of the present embodiment, is mainly achieved through the following technical solutions: comprise the following steps:
Step S100: prepare bacterial classification;
Step S200: cultivate bacterial classification;
Step S300: the collection of inclusion body; By bacterial classification freeze-drying below-20 DEG C of cultivating, then dissolve by buffer A, vibrate under room temperature state 2-4h, and collecting precipitation after solid-liquid separation, obtains inclusion body;
Step S400:RRhPI preliminary purification; After the inclusion body buffer B of collection is dissolved completely, again with buffer B and damping fluid C volume ratio for 1:2-1:10 adds damping fluid C, protein concentration is made to be 0.05-0.5mg/ml, then go up in buffer B balance DEAE-Sepharose FF post, use NaCl gradient elution, collect the elutriant containing RRhPI;
Step S500:RRhPI recombinates renaturation; RRhPI after preliminary purification takes off urea by Sephadex G-25, then is transformed in damping fluid D, places more than 18h, obtains RRhPI renaturation solution for 4 DEG C;
Step S600: enzyme cuts conversion; Be 2:1:2000 by the mass ratio of trypsinase, protaminase, albumen, in RRhPI renaturation solution, add trypsinase and protaminase, room temperature enzyme cuts 50-80min, then by 0.1mol/L ZnCl2 termination reaction and precipitation generates hI;
The purifying of step S700:hI; The purifying of hI comprises thick purifying and consummateization; During described thick purifying, by the two step column chromatographies of the hI in step S600 by the NaCl of Tris-HCl and 0-1.0mmol/L of preset 8-12mmol/L, preliminary purification is carried out to hI and obtains crude product; During described consummateization, carry out refiningly obtaining fine work to crude product with the chromatogram acetonitrile of 10-20mmol/L;
Step S800:hI finished product; Fine work enters filtration sterilization room, less than-20 DEG C freeze-drying again after repeatedly crystallization and washing, obtains hI finished product;
Described step S100 specifically refers to:
Step S110: with human pancreas's cDNA library for template, extracts goal gene by pcr amplification technology;
Step S120: by pQE-40 plasmid that alkaline lysis method of extracting ring fills from the intestinal bacteria cultivated by cell engineering;
Step S130: take out goal gene and pQE-40 plasmid respectively, utilize respectively restriction enzyme Bam HI and Hind III simultaneously enzyme cut and produce sticky end;
Step S140: below 20 DEG C, under the effect of T4DNA ligase enzyme, passes through sticky end by goal gene and pQE-40 Plasmids conjugation complementary, forms the recombinant DNA of an energy expression of insulin;
Step S150: the recombinant DNA containing goal gene is put into colibacillary nutrient solution and adds calcium chloride, makes intestinal bacteria be absorbed by recombinant DNA, recombinant DNA Transformed E .coli competent cell M15;
Described step S200 specifically refers to:
Step S210: take out the bacterial classification prepared in step S100 and activate;
Step S220: adopt second order fermentation mode, constant temperature oscillation 2-3h;
Step S230: induce RRhPI/pQE40 E.coli M15 bacterial classification to express RRhPI with the IPTG of 0.5mmol/L.
Embodiment 2:
The present embodiment does further optimization on the basis of above-described embodiment, further, described step S210 specifically refers to: getting RRhPI/pQE40 E.coli M15 slant strains one ring, to be inoculated in pH value be in the substratum of 6.8-7.6, carry out shaking culture with the speed of 200-280rpm under room temperature condition, air flow is 1:1.3-1:1.6; The main component of described substratum is 3-8g/L Tryptones, 1-3g/L L-glutamic acid, 6-8g/L yeast extract, 0.1-1.0g/L ammonium sulfate, 2-3g/L glucose, 2-3g/L glycerine, 90-110mg/L penbritin, 40-60mg/L kantlex.Other parts of the present embodiment are same as the previously described embodiments, therefore repeat no more.
Embodiment 3:
The present embodiment does further optimization on the basis of above-described embodiment, further, the optimal proportion of described substratum is 5g/L Tryptones, 2g/L L-glutamic acid, 7g/L yeast extract, 0.5g/L ammonium sulfate, 2.5g/L glucose, 2.7g/L glycerine, 100mg/L penbritin, 50mg/L kantlex; Described substratum NaOH regulates pH value to 7.2.Other parts of the present embodiment are same as the previously described embodiments, therefore repeat no more.
Embodiment 4:
The present embodiment does further optimization on the basis of above-described embodiment, and further, in described step S300, the main component of buffer A is 40-60mmol/L Tris-HCl, 0.2-0.8mmol/L EDTA, 40-60mmol/L NaCl, 5% glycerine, 0.1-0.5mmol/L DTT; The pH value of described buffer A is 7.8-8.2.Other parts of the present embodiment are same as the previously described embodiments, therefore repeat no more.
Embodiment 5:
The present embodiment does further optimization on the basis of above-described embodiment, and further, in described step S300, the main component of buffer A is 50mmol/L Tris-HCl, 0.5mmol/L EDTA, 50mmol/L NaCl, 5% glycerine, 0.3mmol/L DTT; The pH value of described buffer A is 7.9.Other parts of the present embodiment are same as the previously described embodiments, therefore repeat no more.
Embodiment 6:
The present embodiment does further optimization on the basis of above-described embodiment, and further, in described step S400, the main component of buffer B is 0.1%-0.3% β-thioglycol, 5-50mmol/L Tris-HCl, 5-10mol/L urea; The pH value of described buffer B is 7.8-8.2.Other parts of the present embodiment are same as the previously described embodiments, therefore repeat no more.
Embodiment 7:
The present embodiment does further optimization on the basis of above-described embodiment, and further, in described step S400, the main component of buffer B is 0.2% β-thioglycol, 30mmol/L Tris-HCl, 8mol/L urea; The pH value of described buffer B is 8.0.Other parts of the present embodiment are same as the previously described embodiments, therefore repeat no more.
Embodiment 8:
The present embodiment does further optimization on the basis of above-described embodiment, and further, in described step S400, the main component of damping fluid C is the halfcystine of 40-70mmol/L Gly-NaOH, 0.3%-0.5%; The pH value of described damping fluid C is 9-10.Other parts of the present embodiment are same as the previously described embodiments, therefore repeat no more.
Embodiment 9:
The present embodiment does further optimization on the basis of above-described embodiment, and further, in described step S500, the main component of damping fluid D is 40-70mmol/L Gly-NaOH, 10-50mmol/L GSSG, 10-50mmol/L GSH; The pH value of described damping fluid D is 9-10.Other parts of the present embodiment are same as the previously described embodiments, therefore repeat no more.
Embodiment 10:
The present embodiment does further optimization on the basis of above-described embodiment, and further, in described step S500, the main component of damping fluid D is 50mmol/L Gly-NaOH, 30mmol/L GSSG, 30mmol/L GSH; The pH value of described damping fluid D is 9.5.Other parts of the present embodiment are same as the previously described embodiments, therefore repeat no more.
Embodiment 11:
A recombinant human insulin's industrial production process, comprises the following steps:
Step S100: prepare bacterial classification; Described step S100 specifically refers to:
Step S110: with human pancreas's cDNA library for template, extracts goal gene by pcr amplification technology;
Step S120: by pQE-40 plasmid that alkaline lysis method of extracting ring fills from the intestinal bacteria cultivated by cell engineering;
Step S130: take out goal gene and pQE-40 plasmid respectively, utilize respectively restriction enzyme Bam HI and Hind III simultaneously enzyme cut and produce sticky end;
Step S140: at 10 DEG C, T
4under the effect of DNA ligase, goal gene and pQE-40 Plasmids conjugation are passed through sticky end complementary, form the recombinant DNA of an energy expression of insulin;
Step S150: the recombinant DNA containing goal gene is put into colibacillary nutrient solution and adds calcium chloride, makes intestinal bacteria be absorbed by recombinant DNA, recombinant DNA Transformed E .coli competent cell M15;
Step S200: cultivate bacterial classification; Described step S200 specifically refers to:
Step S210: take out the bacterial classification prepared in step S100 and activate, specifically refer to and get RRhPI/pQE40 E.coli M15 slant strains one ring to be inoculated in pH value be in the substratum of 7.2, carry out shaking culture with the speed of 250rpm under room temperature condition, air flow is 1:1.5; The main component of described substratum is 6g/L Tryptones, 2g/L L-glutamic acid, 7g/L yeast extract, 0.6g/L ammonium sulfate, 2.5g/L glucose, 2.5g/L glycerine, 100mg/L penbritin, 50mg/L kantlex;
Step S220: adopt second order fermentation mode, constant temperature oscillation 2h;
Step S230: induce RRhPI/pQE40 E.coli M15 bacterial classification to express RRhPI with the IPTG of 0.5mmol/L;
Step S300: the collection of inclusion body; By the bacterial classification of cultivation-25 DEG C of freeze-drying, then dissolve by buffer A, vibrate under room temperature state 3h, and collecting precipitation after solid-liquid separation, obtains inclusion body; In described step S300, the main component of buffer A is 50mmol/L Tris-HCl, 0.5mmol/L EDTA, 50mmol/L NaCl, 5% glycerine, 0.3mmol/L DTT; The pH value of described buffer A is 8.0;
Step S400:RRhPI preliminary purification; After the inclusion body buffer B of collection is dissolved completely, again with buffer B and damping fluid C volume ratio for 1:5 adds damping fluid C, protein concentration is made to be 0.4-0.5mg/ml, then go up in buffer B balance DEAE-Sepharose FF post, use NaCl gradient elution, collect the elutriant containing RRhPI; In described step S400, the main component of buffer B is 0.25% β-thioglycol, 30mmol/L Tris-HCl, 7mol/L urea; The pH value of described buffer B is 8.0; In described step S400, the main component of damping fluid C is 60mmol/L Gly-NaOH, 0.5% halfcystine; The pH value of described damping fluid C is 9.2;
Step S500:RRhPI recombinates renaturation; RRhPI after preliminary purification takes off urea by Sephadex G-25, then is transformed in damping fluid D, places 24h, obtains RRhPI renaturation solution for 4 DEG C; In described step S500, the main component of damping fluid D is 60mmol/L Gly-NaOH, 30mmol/L GSSG, 30mmol/L GSH; The pH value of described damping fluid D is 9.2;
Step S600: enzyme cuts conversion; Be 2:1:2000 by the mass ratio of trypsinase, protaminase, albumen, in RRhPI renaturation solution, add trypsinase and protaminase, room temperature enzyme cuts 80min, then by 0.1mol/L ZnCl2 termination reaction and precipitation generates hI;
The purifying of step S700:hI; The purifying of hI comprises thick purifying and consummateization; During described thick purifying, by the two step column chromatographies of the hI in step S600 by the NaCl of Tris-HCl and 0.6mmol/L of preset 10mmol/L, preliminary purification is carried out to hI and obtains crude product; During described consummateization, carry out refiningly obtaining fine work to crude product with the chromatogram acetonitrile of 10-20mmol/L;
Step S800:hI finished product; Fine work enters filtration sterilization room ,-25 DEG C of freeze-drying again after repeatedly crystallization and washing, obtains hI finished product.
Other parts of the present embodiment are same as the previously described embodiments, therefore repeat no more.
Embodiment 12:
A recombinant human insulin's industrial production process, comprises the following steps:
Step S100: prepare bacterial classification; Described step S100 specifically refers to:
Step S110: with human pancreas's cDNA library for template, extracts goal gene by pcr amplification technology;
Step S120: by pQE-40 plasmid that alkaline lysis method of extracting ring fills from the intestinal bacteria cultivated by cell engineering;
Step S130: take out goal gene and pQE-40 plasmid respectively, utilize respectively restriction enzyme Bam HI and Hind III simultaneously enzyme cut and produce sticky end;
Step S140: at 10 DEG C, T
4under the effect of DNA ligase, goal gene and pQE-40 Plasmids conjugation are passed through sticky end complementary, form the recombinant DNA of an energy expression of insulin;
Step S150: the recombinant DNA containing goal gene is put into colibacillary nutrient solution and adds calcium chloride, makes intestinal bacteria be absorbed by recombinant DNA, recombinant DNA Transformed E .coli competent cell M15;
Step S200: cultivate bacterial classification; Described step S200 specifically refers to:
Step S210: take out the bacterial classification prepared in step S100 and activate, specifically refer to and get RRhPI/pQE40 E.coli M15 slant strains one ring to be inoculated in pH value be in the substratum of 7.2, carry out shaking culture with the speed of 280rpm under room temperature condition, air flow is 1:1.6; The main component of described substratum is 8g/L Tryptones, 3g/L L-glutamic acid, 8g/L yeast extract, 1.0g/L ammonium sulfate, 3g/L glucose, 3g/L glycerine, 110mg/L penbritin, 60mg/L kantlex;
Step S220: adopt second order fermentation mode, constant temperature oscillation 3h;
Step S230: induce RRhPI/pQE40 E.coli M15 bacterial classification to express RRhPI with the IPTG of 0.5mmol/L;
Step S300: the collection of inclusion body; By the bacterial classification of cultivation-25 DEG C of freeze-drying, then dissolve by buffer A, vibrate under room temperature state 3h, and collecting precipitation after solid-liquid separation, obtains inclusion body; In described step S300, the main component of buffer A is 60mmol/L Tris-HCl, 0.8mmol/L EDTA, 60mmol/L NaCl, 5% glycerine, 0.5mmol/L DTT; The pH value of described buffer A is 8.1;
Step S400:RRhPI preliminary purification; After the inclusion body buffer B of collection is dissolved completely, again with buffer B and damping fluid C volume ratio for 1:8 adds damping fluid C, protein concentration is made to be 0.4-0.5mg/ml, then go up in buffer B balance DEAE-Sepharose FF post, use NaCl gradient elution, collect the elutriant containing RRhPI; In described step S400, the main component of buffer B is 0.3% β-thioglycol, 45mmol/L Tris-HCl, 10mol/L urea; The pH value of described buffer B is 8.1; In described step S400, the main component of damping fluid C is 70mmol/L Gly-NaOH, 0.5% halfcystine; The pH value of described damping fluid C is 9.4;
Step S500:RRhPI recombinates renaturation; RRhPI after preliminary purification takes off urea by Sephadex G-25, then is transformed in damping fluid D, places 24h, obtains RRhPI renaturation solution for 4 DEG C; In described step S500, the main component of damping fluid D is 70mmol/L Gly-NaOH, 45mmol/L GSSG, 45mmol/L GSH; The pH value of described damping fluid D is 9.4;
Step S600: enzyme cuts conversion; Be 2:1:2000 by the mass ratio of trypsinase, protaminase, albumen, in RRhPI renaturation solution, add trypsinase and protaminase, room temperature enzyme cuts 80min, then by 0.1mol/L ZnCl2 termination reaction and precipitation generates hI;
The purifying of step S700:hI; The purifying of hI comprises thick purifying and consummateization; During described thick purifying, by the two step column chromatographies of the hI in step S600 by the NaCl of Tris-HCl and 0.6mmol/L of preset 10mmol/L, preliminary purification is carried out to hI and obtains crude product; During described consummateization, carry out refiningly obtaining fine work to crude product with the chromatogram acetonitrile of 10-20mmol/L;
Step S800:hI finished product; Fine work enters filtration sterilization room ,-25 DEG C of freeze-drying again after repeatedly crystallization and washing, obtains hI finished product.
Other parts of the present embodiment are same as the previously described embodiments, therefore repeat no more.
Embodiment 13:
A recombinant human insulin's industrial production process, comprises the following steps:
Step S100: prepare bacterial classification; Described step S100 specifically refers to:
Step S110: with human pancreas's cDNA library for template, extracts goal gene by pcr amplification technology;
Step S120: by pQE-40 plasmid that alkaline lysis method of extracting ring fills from the intestinal bacteria cultivated by cell engineering;
Step S130: take out goal gene and pQE-40 plasmid respectively, utilize respectively restriction enzyme Bam HI and Hind III simultaneously enzyme cut and produce sticky end;
Step S140: at 10 DEG C, T
4under the effect of DNA ligase, goal gene and pQE-40 Plasmids conjugation are passed through sticky end complementary, form the recombinant DNA of an energy expression of insulin;
Step S150: the recombinant DNA containing goal gene is put into colibacillary nutrient solution and adds calcium chloride, makes intestinal bacteria be absorbed by recombinant DNA, recombinant DNA Transformed E .coli competent cell M15;
Step S200: cultivate bacterial classification; Described step S200 specifically refers to:
Step S210: take out the bacterial classification prepared in step S100 and activate, specifically refer to and get RRhPI/pQE40 E.coli M15 slant strains one ring to be inoculated in pH value be in the substratum of 7.2, carry out shaking culture with the speed of 250rpm under room temperature condition, air flow is 1:1.5; The main component of described substratum is 5g/L Tryptones, 2g/L L-glutamic acid, 7g/L yeast extract, 0.5g/L ammonium sulfate, 2.5g/L glucose, 2.7g/L glycerine, 100mg/L penbritin, 50mg/L kantlex;
Step S220: adopt second order fermentation mode, constant temperature oscillation 2.5h;
Step S230: induce RRhPI/pQE40 E.coli M15 bacterial classification to express RRhPI with the IPTG of 0.5mmol/L;
Step S300: the collection of inclusion body; By the bacterial classification of cultivation-25 DEG C of freeze-drying, then dissolve by buffer A, vibrate under room temperature state 3h, and collecting precipitation after solid-liquid separation, obtains inclusion body; In described step S300, the main component of buffer A is 50mmol/L Tris-HCl, 0.5mmol/L EDTA, 50mmol/L NaCl, 5% glycerine, 0.3mmol/L DTT; The pH value of described buffer A is 8.0;
Step S400:RRhPI preliminary purification; After the inclusion body buffer B of collection is dissolved completely, again with buffer B and damping fluid C volume ratio for 1:5 adds damping fluid C, protein concentration is made to be 0.4-0.5mg/ml, then go up in buffer B balance DEAE-Sepharose FF post, use NaCl gradient elution, collect the elutriant containing RRhPI; In described step S400, the main component of buffer B is 0.2% β-thioglycol, 30mmol/L Tris-HCl, 8mol/L urea; The pH value of described buffer B is 8.0; In described step S400, the main component of damping fluid C is 55mmol/L Gly-NaOH, 0.4% halfcystine; The pH value of described damping fluid C is 9.2;
Step S500:RRhPI recombinates renaturation; RRhPI after preliminary purification takes off urea by Sephadex G-25, then is transformed in damping fluid D, places 24h, obtains RRhPI renaturation solution for 4 DEG C; In described step S500, the main component of damping fluid D is 55mmol/L Gly-NaOH, 40mmol/L GSSG, 40mmol/L GSH; The pH value of described damping fluid D is 9.2;
Step S600: enzyme cuts conversion; Be 2:1:2000 by the mass ratio of trypsinase, protaminase, albumen, in RRhPI renaturation solution, add trypsinase and protaminase, room temperature enzyme cuts 80min, then by 0.1mol/L ZnCl2 termination reaction and precipitation generates hI;
The purifying of step S700:hI; The purifying of hI comprises thick purifying and consummateization; During described thick purifying, by the two step column chromatographies of the hI in step S600 by the NaCl of Tris-HCl and 0.6mmol/L of preset 10mmol/L, preliminary purification is carried out to hI and obtains crude product; During described consummateization, carry out refiningly obtaining fine work to crude product with the chromatogram acetonitrile of 10-20mmol/L;
Step S800:hI finished product; Fine work enters filtration sterilization room ,-25 DEG C of freeze-drying again after repeatedly crystallization and washing, obtains hI finished product.
The above is only preferred embodiment of the present invention, and not do any pro forma restriction to the present invention, every any simple modification, equivalent variations done above embodiment according to technical spirit of the present invention, all falls within protection scope of the present invention.
Claims (10)
1. a recombinant human insulin's industrial production process, is characterized in that, comprises the following steps:
Step S100: prepare bacterial classification;
Step S200: cultivate bacterial classification;
Step S300: the collection of inclusion body; By bacterial classification freeze-drying below-20 DEG C of cultivating, then dissolve by buffer A, vibrate under room temperature state 2-4h, and collecting precipitation after solid-liquid separation, obtains inclusion body;
Step S400:RRhPI preliminary purification; After the inclusion body buffer B of collection is dissolved completely, again with buffer B and damping fluid C volume ratio for 1:2-1:10 adds damping fluid C, protein concentration is made to be 0.05-0.5mg/ml, then go up in buffer B balance DEAE-Sepharose FF post, use NaCl gradient elution, collect the elutriant containing RRhPI;
Step S500:RRhPI recombinates renaturation; RRhPI after preliminary purification takes off urea by Sephadex G-25, then is transformed in damping fluid D, places more than 18h, obtains RRhPI renaturation solution for 4 DEG C;
Step S600: enzyme cuts conversion; Be 2:1:2000 by the mass ratio of trypsinase, protaminase, albumen, in RRhPI renaturation solution, add trypsinase and protaminase, room temperature enzyme cuts 50-80min, then uses 0.1mol/L ZnCl
2termination reaction also precipitates generation hI;
The purifying of step S700:hI; The purifying of hI comprises thick purifying and consummateization; During described thick purifying, by the two step column chromatographies of the hI in step S600 by the NaCl of Tris-HCl and 0-1.0mmol/L of preset 8-12mmol/L, preliminary purification is carried out to hI and obtains crude product; During described consummateization, carry out refiningly obtaining fine work to crude product with the chromatogram acetonitrile of 10-20mmol/L;
Step S800:hI finished product; Fine work enters filtration sterilization room, less than-20 DEG C freeze-drying again after repeatedly crystallization and washing, obtains hI finished product;
Described step S100 specifically refers to:
Step S110: with human pancreas's cDNA library for template, extracts goal gene by pcr amplification technology;
Step S120: by pQE-40 plasmid that alkaline lysis method of extracting ring fills from the intestinal bacteria cultivated by cell engineering;
Step S130: take out goal gene and pQE-40 plasmid respectively, utilize respectively restriction enzyme Bam HI and Hind III simultaneously enzyme cut and produce sticky end;
Step S140: below 20 DEG C, T
4under the effect of DNA ligase, goal gene and pQE-40 Plasmids conjugation are passed through sticky end complementary, form the recombinant DNA of an energy expression of insulin;
Step S150: the recombinant DNA containing goal gene is put into colibacillary nutrient solution and adds calcium chloride, makes intestinal bacteria be absorbed by recombinant DNA, recombinant DNA Transformed E .coli competent cell M15;
Described step S200 specifically refers to:
Step S210: take out the bacterial classification prepared in step S100 and activate;
Step S220: adopt second order fermentation mode, constant temperature oscillation 2-3h;
Step S230: induce RRhPI/pQE40 E.coli M15 bacterial classification to express RRhPI with the IPTG of 0.5mmol/L.
2. the industrial production process of a kind of recombinant human insulin according to claim 1, it is characterized in that: described step S210 specifically refers to: getting RRhPI/pQE40 E.coli M15 slant strains one ring, to be inoculated in pH value be in the substratum of 6.8-7.6, carry out shaking culture with the speed of 200-280rpm under room temperature condition, air flow is 1:1.3-1:1.6; The main component of described substratum is 3-8g/L Tryptones, 1-3g/L L-glutamic acid, 6-8g/L yeast extract, 0.1-1.0g/L ammonium sulfate, 2-3g/L glucose, 2-3g/L glycerine, 90-110mg/L penbritin, 40-60mg/L kantlex.
3. the industrial production process of a kind of recombinant human insulin according to claim 2, it is characterized in that: the optimal proportion of described substratum is 5g/L Tryptones, 2g/L L-glutamic acid, 7g/L yeast extract, 0.5g/L ammonium sulfate, 2.5g/L glucose, 2.7g/L glycerine, 100mg/L penbritin, 50mg/L kantlex; Described substratum NaOH regulates pH value to 7.2.
4. the industrial production process of a kind of recombinant human insulin according to claim 1, it is characterized in that: in described step S300, the main component of buffer A is 40-60mmol/L Tris-HCl, 0.2-0.8mmol/L EDTA, 40-60mmol/L NaCl, 5% glycerine, 0.1-0.5mmol/L DTT; The pH value of described buffer A is 7.8-8.2.
5. the industrial production process of a kind of recombinant human insulin according to claim 4, it is characterized in that: in described step S300, the main component of buffer A is 50mmol/L Tris-HCl, 0.5mmol/L EDTA, 50mmol/L NaCl, 5% glycerine, 0.3mmol/L DTT; The pH value of described buffer A is 7.9.
6. the industrial production process of a kind of recombinant human insulin according to claim 1, is characterized in that: in described step S400, the main component of buffer B is 0.1%-0.3% β-thioglycol, 5-50mmol/L Tris-HCl, 5-10mol/L urea; The pH value of described buffer B is 7.8-8.2.
7. the industrial production process of a kind of recombinant human insulin according to claim 6, is characterized in that: in described step S400, the main component of buffer B is 0.2% β-thioglycol, 30mmol/L Tris-HCl, 8mol/L urea; The pH value of described buffer B is 8.0.
8. the industrial production process of a kind of recombinant human insulin according to claim 1, is characterized in that: in described step S400, the main component of damping fluid C is 40-70mmol/L Gly-NaOH, 0.3%-0.5% halfcystine; The pH value of described damping fluid C is 9-10.
9. the industrial production process of a kind of recombinant human insulin according to claim 1, is characterized in that: in described step S500, the main component of damping fluid D is 40-70mmol/L Gly-NaOH, 10-50mmol/L GSSG, 10-50mmol/L GSH; The pH value of described damping fluid D is 9-10.
10. the industrial production process of a kind of recombinant human insulin according to claim 7, is characterized in that: in described step S500, the main component of damping fluid D is 50mmol/L Gly-NaOH, 30mmol/L GSSG, 30mmol/L GSH; The pH value of described damping fluid D is 9.5.
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| EP0434605A2 (en) * | 1989-11-29 | 1991-06-26 | Ciba-Geigy Ag | Novel process for the production of unfused protein in E. coli |
| US5986048A (en) * | 1997-08-18 | 1999-11-16 | Hoechst Marion Roussel Deutschland | Process for obtaining insulin precursors having correctly bonded cystine bridges |
| CN101173006A (en) * | 2006-10-30 | 2008-05-07 | 江苏万邦生化医药股份有限公司 | Method for producing recombined insulin human |
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| EP0434605A2 (en) * | 1989-11-29 | 1991-06-26 | Ciba-Geigy Ag | Novel process for the production of unfused protein in E. coli |
| US5986048A (en) * | 1997-08-18 | 1999-11-16 | Hoechst Marion Roussel Deutschland | Process for obtaining insulin precursors having correctly bonded cystine bridges |
| CN101173006A (en) * | 2006-10-30 | 2008-05-07 | 江苏万邦生化医药股份有限公司 | Method for producing recombined insulin human |
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