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Fermenting technology for improving expression amount of fusion protein of recombinant human brain natriuretic peptide

A technology of human brain natriuretic peptide and fermentation process, which is applied in the field of fermentation process to improve the expression of recombinant human brain natriuretic peptide fusion protein, which can solve the problem that industrial production cannot be realized, it has no commercial development value, and the expression of fusion protein is affected etc. to achieve good commercial development value, low production cost, and easy operation and control

Active Publication Date: 2017-09-19
西藏诺迪康药业股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, recombinant human brain natriuretic peptide is mainly prepared through gene recombination technology, which uses rhBNP genetically engineered bacteria to ferment and cultivate, and then, separates and purifies, prepares, and finally obtains recombinant human brain natriuretic peptide products; among them, the fermentation process is the decision One of the key steps in the production of recombinant human brain natriuretic peptide, which directly affects the expression level of the fusion protein. If the protein expression level is too low, it will lead to a substantial increase in the production cost of the unit product, which is considered not to be commercially exploitable. value, and thus cannot realize industrialized production

Method used

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  • Fermenting technology for improving expression amount of fusion protein of recombinant human brain natriuretic peptide

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Effect test

Embodiment 1

[0031] In the fermentor, add 18.5L of BHD medium and 74mL of glycerol, and ampicillin to its final concentration of 100mg / L, inoculate 400mL of recombinant human brain natriuretic peptide (rhBNP) genetically engineered bacteria, adjust the ventilation rate so that the Dissolved oxygen (ie D.O. value) is 50%, add hydrochloric acid or sodium hydroxide to adjust the pH value to 7.0, and the culture temperature is 37°C; when OD 600 (referring to the absorbance value at 600nm wavelength place) when being 12, adding IPTG (isopropyl thiogalactopyranoside) to its final concentration is 0.5mmol / L (mmol / L refers to millimole / liter) induction, and Add 200mL / h feed solution A to the fermenter, stop the fermentation culture after 4h, and harvest the bacteria;

[0032] in,

[0033] The formula of the BHD medium is as follows: each liter of BHD medium contains 5 g of tryptone (Tryptone), 10 g of yeast powder (Yeast Extract), potassium dihydrogen phosphate (KH 2 PO 4 ) 2.31g, dipotassium h...

Embodiment 2

[0039] In the fermentor, add 20L of BHD medium, 68mL of glycerol, and ampicillin to a final concentration of 100mg / L, inoculate 400mL of recombinant human brain natriuretic peptide (rhBNP) genetically engineered bacteria, adjust the aeration rate to make the solution in the fermentation broth Oxygen content (being D.O. value) is 30%, adds hydrochloric acid or sodium hydroxide to adjust pH value to be 7.0, and culture temperature is 37 ℃; When OD 600 At 12 o'clock, add IPTG (isopropylthiogalactopyranoside) to its final concentration of 1.0mmol / L induction, and add 200mL / h feed solution A to the fermenter, stop fermentation after 4h, and harvest bacteria;

[0040] in,

[0041]The formula of the BHD medium is as follows: each liter of BHD medium contains 5 g of tryptone (Tryptone), 10 g of yeast powder (Yeast Extract), potassium dihydrogen phosphate (KH 2 PO 4 ) 2.31g, dipotassium hydrogen phosphate (K 2 HPO 4 ) 12.54g, magnesium sulfate (MgSO 4 ) 0.5g, sodium chloride (NaC...

Embodiment 3

[0045] In the fermentor, add 18.5L of BHD medium and 74mL of glycerol, and ampicillin to its final concentration of 100mg / L, inoculate 400mL of recombinant human brain natriuretic peptide (rhBNP) genetically engineered bacteria, adjust the ventilation rate so that the Dissolved oxygen (ie D.O. value) is 50%, add hydrochloric acid or sodium hydroxide to adjust the pH value to 7.0, and the culture temperature is 37°C; when OD 600 At 12 o'clock, add IPTG (isopropylthiogalactopyranoside) to its final concentration of 0.8mmol / L induction, and feed 200mL / h feed solution A in the fermentor, stop fermentation after 4h, harvest bacteria;

[0046] in,

[0047] The formula of the BHD medium is as follows: each liter of BHD medium contains 5 g of tryptone (Tryptone), 10 g of yeast powder (Yeast Extract), potassium dihydrogen phosphate (KH 2 PO 4 ) 2.31g, dipotassium hydrogen phosphate (K 2 HPO 4 ) 12.54g, magnesium sulfate (MgSO 4 ) 0.5g, sodium chloride (NaCl) 0.6g, vitamins (panto...

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Abstract

The invention discloses a fermenting technology for improving expression amount of fusion protein of recombinant human brain natriuretic peptide. The fermenting technology comprises the following steps of adding 18 to 20L of BHD culture medium, 65 to 80mL of glycerin, and ampicillin into a fermenting tank until the final concentration is 100mg / L; inoculating, adjusting the air ventilation amount until the dissolved oxygen content in the fermenting liquid is 30% to 50%, adjusting the pH (potential of hydrogen) value to 6.8 to 7.2, and culturing at the temperature of 37 DEG C; when OD600 is 10 to 15, adding isopropyl thiogalactoside until the final concentration is 0.5 to 1.0mmol / L, inducing, adding 150 to 300mL / A of supplementing liquid A into the fermenting tank, stopping fermenting culture after 3 to 6h, and harvesting bacteria. The fermenting technology has the advantages that the expression level of rhBNP fusion protein is obviously improved, the expression amount reaches up to 34.5% to 38.3%, and the good commercial development value and great success in commerce are obtained.

Description

technical field [0001] The invention belongs to the field of biological fermentation, and in particular relates to a fermentation process for increasing the expression of recombinant human brain natriuretic peptide fusion protein. Background technique [0002] Heart failure is a major public health problem. Studies have shown that the prevalence of people over the age of 35 in China is as high as 2%, with about 10 million patients, and 500,000 new patients every year, which seriously threatens the life and health of our people. With the aging of the population, the number of heart failure patients in my country will further increase. [0003] Recombinant human brain natriuretic peptide (rhBNP, trade name: Xinhuosu) can effectively treat heart failure and other diseases, quickly relieve patients with dyspnea, diuresis and natriuresis, antagonize neuroendocrine, and resist heart remodeling, and comprehensively protect the heart It has been included and recommended in the "Guid...

Claims

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Application Information

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IPC IPC(8): C12P21/02
CPCC07K14/58C07K2319/00
Inventor 张艳任东升刘希望
Owner 西藏诺迪康药业股份有限公司
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