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Method for preparing human brain natriuretic peptide by genetic engineering recombination technology

A technology of human brain natriuretic peptide and recombinant technology, which is applied in the field of human brain natriuretic peptide preparation by recombinant technology, can solve the problems of high cost, low recovery rate, complicated production process, etc. simple effect

Inactive Publication Date: 2005-03-16
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The purpose of the present invention is to provide a method for preparing human brain natriuretic peptide by novel genetic engineering technology in order to overcome the shortcomings of the current Escherichia coli recombinant expression of human brain natriuretic peptide.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] CKS mut Construction of / BNP fusion gene

[0020] The following experimental methods refer to "Molecular Cloning Experiment Guide" translated by Jin Dongyan et al. (1995, Science Press). The CKS gene was cloned from the E.coli K12 strain by the Microbial Genetic Engineering Laboratory of the School of Life Sciences, Shenzhen University according to the genebank sequence. After sequence analysis, it was consistent with the Kds B gene sequence of the E.coli K12 strain reported by Genebank. Mutate the last Met codon ATG at the C-terminus of CKS to Thr codon ATC by site-directed mutagenesis to obtain CKS mut Gene. The 32 amino acid coding regions of the BNP active site were selected as follows:

[0021] Ser Pro Lys Met Val Glu Gly Ser Gly Cys Phe Gly Arg Lys MetAsp Arg Ile Ser Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg ArgHis

[0022] The coding region gene was synthesized according to common yeast codons. CKS mut The artificially synthesized gene and BNP active ...

Embodiment 2

[0024] CKS mut Construction of Yeast Expression of / BNP Fusion Gene and Screening of Engineering Yeast

[0025] PCR was used to convert the above CKS mut EcoRI was added to the 5' end of the / BNP fusion gene, and a NotI site was added to the 3', and cloned into the Pichia secretion expression vector pPIC9 EcoRI-Not I of methanol yeast, to construct the secretion expression vector pPIC CKS / BNP of methanol yeast (see Jin Dongyan et al. Cloning Experiment Guide "1995, Science Press).

[0026]Yeast transformation was carried out according to the method of Faber (1994). Take a single colony of GS115 and inoculate it in 2.0ml YEPD, culture overnight at 28-30°C, then transfer to 200ml YEPD for 5 hours, collect by centrifugation at 6000g×5min, suspend the bacteria in 25mM sodium phosphate buffer (pH7.0) containing 25mM DTT ), placed at 30°C for 15 minutes, collected by centrifugation, and washed twice with 200ml of 10mM Tris-Cl (STM, pH 7.0) containing 270mM sucrose, and the obtain...

Embodiment 3

[0029] With CKS mut / BNP Fusion Gene Engineering Yeast Fermentation and Product Purification

[0030] Growth medium (g / L): yeast nitrogen source 13.4, biotin 4×10 -4 , peptone 20, yeast extract 10, pH 6.0, fermentation medium (g / L): yeast nitrogen source 13.4, biotin 4×10 -4 , peptone 20, yeast extract 10, trace element mixture 5ml.

[0031] 100ml of growth medium was placed in a 500ml Erlenmeyer flask, and cultured with shaking at 200r / min at 28°C after inoculation. After the OD600 of the seed liquid reaches 4.0, it is replanted into a 5-liter self-controlled fermenter for fermentation. Fermentation tank adopts fermentation medium, 200-300r / min, cultured at 28°C, and the pH value is controlled at 6.0 with ammonia water. Monitor the growth of the bacteria by observing the dissolved oxygen, and control the dissolved oxygen between 25%. When the dissolved oxygen rises, add 2% glycerol, stop adding glycerin after about 48 hours of cultivation, let the bacteria consume the re...

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Abstract

The invention relates to a nesiritide preparation method using gene engineering technology. The method includes: artificial synthesis of corresponding gene of human nesiritide BNP active position 32 amino acid, combining it with 3'end of mutated CMP-3-deoxy-D-manna-octulosonic acid synthase gene, in the recombinant gene the enterokinase identification cutting point is contained, cloning the recombinant gene into methanol nutrition type yeast Pichia expression vector, thus obtaining recombinant gene highly expressed engineered yeast, fermenting the engineered yeast ,extracting , proceeding chromatography and purifying to produce recombinant protein, cutting with enterokinase, separating and purifying, obtaining recombinant human nesiritide. The recombinant peptide has huaman nesiritide activity after in vitro experiment.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for preparing human brain natriuretic peptide by recombinant technology. Background technique [0002] Cardiovascular and cerebrovascular diseases are recognized as the "first killer" of human health in the world. Cardiovascular diseases account for about 20% of all deaths in my country every year. There are many kinds of cardiovascular and cerebrovascular diseases, and heart failure is the final stage of many heart diseases. [0003] Heart failure is a group of clinically more common cardiovascular syndromes, and it is an almost inevitable outcome for most patients with structural heart disease. Hypertensive heart disease, coronary heart disease, myocardial infarction, rheumatic heart disease, cor pulmonale, dilated cardiomyopathy, hypertrophic cardiomyopathy, myocarditis, tachycardia cardiomyopathy, diabetic cardiomyopathy, hyperthyroid heart disease , Alcoholic c...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/52C12N15/62C12N15/81C12P21/02
Inventor 刘志刚吉坤美喻海琼高波张红云
Owner SHENZHEN UNIV
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