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Human NT-proBNP preparation capable of stable preservation and preparation method thereof

A nt-probnp, stable technology, applied in the field of medicine and biology, can solve the problems of high production conditions, huge investment in eukaryotic expression system, long expression cycle, etc., and achieve the effect of high expression yield, strong stability and high activity

Inactive Publication Date: 2013-01-16
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the eukaryotic expression system requires a huge investment, a long expression cycle, and high production conditions

Method used

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  • Human NT-proBNP preparation capable of stable preservation and preparation method thereof
  • Human NT-proBNP preparation capable of stable preservation and preparation method thereof
  • Human NT-proBNP preparation capable of stable preservation and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Synthesis, cloning, transformation, and double-enzyme digestion identification of the target gene

[0057] The optimized nucleotide sequence encoding human NT-proBNP as described above was synthesized by Shanghai Genomics Bioengineering Technology Service Co., Ltd. After the nucleotide sequence was synthesized, the sequence was cloned into the pET32a vector.

[0058] Transformation of BL21 (DE3) expression strain: Take out the competent cells from the -80°C refrigerator and place them on ice to thaw. After thawing, add 1μg of the synthesized plasmid, ice bath for 30min, heat shock at 42°C for 60-90s, place on ice for 2min (do not move during this process), add 900ul LB medium, shake at 37°C and 160rpm for 45min, take out 100μl and spread on Ampicillin-resistant LB plates were cultured upside down at 37°C overnight. After the growth of monoclonal colonies, culture the monoclonal colonies and preserve the strains.

[0059] Double enzyme digestion identification...

Embodiment 2

[0060] Example 2 Optimization of fermentation conditions of BL21 (DE3) strain transformed with NT-proBNP

[0061] Determination of the growth curve of the strain: use 1% and 0.1% glycerol bacteria in the LB medium respectively, and measure the OD at intervals 600 , respectively to make growth curves to determine the appropriate inoculum size. Finally, 0.1% was selected as the optimal inoculum volume, and the seeds were cultured with shaking overnight.

[0062] Determination of carbon source: 0.1% of glycerol bacteria were inoculated into LB medium (Amp100μg / ml), shaken at 37°C and 210rpm overnight. Inoculate 1% of the inoculum into 15ml medium (on the basis of 2YT medium, respectively take two concentrations of sucrose, glucose, and glycerol, two concentrations of 0.5% and 1%, Amp100μg / ml, and culture on a shaking table at 37°C , 220rpm. Cultivate until OD600>0.8 (about 3-4h), add 1mM IPTG to induce. After 4h, collect the bacteria to measure the OD value of the collected bac...

Embodiment 3

[0072] Expression and purification of embodiment three recombinant human NT-proBNP proteins

[0073] The expression strain BL21 (DE3) transformed with the vector pET32a containing the human NT-proBNP sequence was cultivated using the optimized culture conditions screened in Example 2. Inoculate with 1% inoculum during the cultivation process, use 1% glycerol as fermentation carbon source, nitrogen source is 0.5% yeast powder, 0.5% peptone, 1% NH 4 Cl, the inorganic salt additive is 5mM magnesium sulfate, and the bacteria collection time is 6 hours. After expression, the strain was homogeneously broken and centrifuged under high pressure. The results of electrophoresis showed that the combination of the strain and the vector achieved efficient and soluble expression of the target protein. The results are as follows: Image 6 shown.

[0074] The loading order of the electrophoresis results is from left to right: supernatant of crushed cells, crushed cells of precipitates, tota...

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Abstract

The invention relates to a human NT-proBNP preparation capable of stable preservation, a preparation method thereof, and a method for high-yield recombinant expression of human NT-proBNP; particularly, an escherichia coli expression system is used to produce a recombinant human B-type natriuretic peptide precursor (NT-proBNP), and the preparation process and conditions are optimized to obtain a preparation process for high-yield expression of human NT-proBNP; and the human NT-proBNP preparation capable of stable preservation is obtained by adding a stabilizing protective agent. The natural human NT-proBNP preparation prepared by adding the stabilizing protective agent in the invention can exist stably under conditions of -20 DEG C and -80 DEG C, and the biological activity is not decreased. A stable raw material source is provided for the preparation of NT-proBNP as a clinical diagnosis reagent standard substance and an antibody.

Description

【Technical field】 [0001] The invention relates to a stably preserved human NT-proBNP preparation and a preparation method thereof, as well as a method capable of high-yield recombinant expression of human NT-proBNP, belonging to the field of medical biotechnology. 【Background technique】 [0002] The natriuretic peptide (NP) family is now receiving more and more attention. Most of the human natriuretic peptide family is secreted by the heart and plays a role in controlling water-salt balance and maintaining pressure regulation homeostasis. Cardiovascular natriuretic peptide endocrine components include ANP, BNP, CNP, DNP and VNP. Although the types are different, the evolution of most types of natriuretic peptides in the same species is quite conservative. Only BNP and its amino-terminal precursor BNP (NT-proBNP) differ within species (in mammals). [0003] In addition to the functions shared by natriuretic peptides, B-type natriuretic peptides may also have the effects of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/74G01N33/68C12N15/70
Inventor 许秀丽田亚平刘培郝庆钦
Owner GENERAL HOSPITAL OF PLA
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