Long-acting recombinant human brain natriuretic peptide fusion protein and preparation method and thereof and application

A fusion protein and peptide linker technology, which is applied in the fields of molecular biology and medicine, can solve the problems that affect the wide application of products, affect the drug compliance of patients, and short half-life, so as to improve the purity and activity, ensure safety, and high expression yield Effect

Active Publication Date: 2015-08-26
CHENGDU JINKAI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the short half-life of rhBNP in clinical use (only 18 minutes), frequent administration is required to achieve the therapeutic

Method used

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  • Long-acting recombinant human brain natriuretic peptide fusion protein and preparation method and thereof and application
  • Long-acting recombinant human brain natriuretic peptide fusion protein and preparation method and thereof and application
  • Long-acting recombinant human brain natriuretic peptide fusion protein and preparation method and thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1. Construction of gene expression plasmids encoding recombinant hBNP-Fc fusion proteins

[0057] (1) Preparation of hBNP gene sequence

[0058] According to the gene sequence of hBNP (Genebank accession number: U34833.1), the gene sequence of the leader peptide and mature protein coding region of hBNP was prepared by artificial synthesis, and the synthetic hBNP gene fragment was inserted into the EcoRV restriction in the transfer vector PUC57. In the restriction site, the phBNP plasmid was obtained, and the correctness of the hBNP gene sequence was verified by DNA sequencing.

[0059] (2) Preparation of L-vIgG1Fc fusion gene sequence

[0060] The fusion gene L-vIgG4Fc containing the coding peptide linker (Linker, referred to as L) and the human IgG4Fc variant fragment containing the 5' end BamH I and the 3' end EcoR I restriction endonuclease sites were synthesized by artificial synthesis method. The prepared fusion gene fragment was inserted into the BamH I ...

Embodiment 2

[0063] Example 2. Expression of recombinant hBNP-Fc fusion protein in mammalian cells

[0064] The expression plasmid pCDNA-hBNP-Fc constructed in Example 1 and the plasmid pSV2-dhfr (ATCC) containing the DHFR transcription unit were co-transfected into DHFR enzyme-deficient CHO cells (CHO-DHFR - ). Specific steps are as follows:

[0065] Transfection was carried out by electroporation transfection, using a Gene Pulser Electroporator (Bio-Rad Laboratories, Hercules, CA) with a capacity of 900 μFd, the electric field was set at 240 V, and 5 × 10 cells in a cuvette were used. 7 10 μg pCDNA-hBNP-Fc plasmid DNA linearized with Pvu I and 10 μg pSV2-dhfr were added to each cell for co-transfection. After 24 hours of transfection, the culture medium was changed to a growth medium containing 100 μg / mL G418 and 20 nM MTX, and the clones were cloned in a 96-well cell culture plate by limiting dilution to obtain positive clones that had passed the initial resistance screening. The tra...

Embodiment 3

[0067] Example 3. Separation and purification of recombinant hBNP-Fc fusion protein

[0068] (1) Expanded production of CHO cells highly expressing recombinant hBNP-Fc fusion protein

[0069] After the CHO cell line with stable and high expression in Example 2 was fully adapted to serum-free suspension culture, the cells were transferred to a 5L bioreactor for scale-up culture. Select EX-CELL TM 302 serum-free medium (Sigma, 24326C-100L) was used as the basal medium.

[0070] Add ultra-filtered plant peptone (Kerry, HyPep 7504) at a final concentration of 6 g / L or nutrient supplement (Hyclone, Cell Boost 6) at a final concentration of 8 g / L in the basal medium. After 12 days of culture, the expression level of recombinant hBNP-Fc fusion protein accumulated to 1.85g / L.

[0071] (2) Purification of recombinant hBNP-Fc fusion protein;

[0072] a) clarification: adopt the DOHC and B1HC depth filters of Millipore Company to filter the cell culture fluid obtained by the above-m...

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Abstract

The invention discloses long-acting recombinant human brain natriuretic peptide fusion protein (hBNP-Fc) and a preparation method thereof. An amino acid sequence of the fusion protein sequentially comprises human BNP (hBNP), peptide connectors and human IgG4Fc variants from N to C. The fusion protein has a biological activity similar to or higher than that of rhBNP, a longer half-life period and fewer side effects. The invention also relates to the application of a recombinant hBNP-Fc fusion protein composition in the preparation of acute heart failure treatment and/or prevention medicine.

Description

technical field [0001] The invention relates to the fields of molecular biology and medicine. More specifically, the present invention relates to a long-acting recombinant human brain natriuretic peptide fusion protein and its preparation method and application. Background technique [0002] With the acceleration of the aging process of the population and the increase in the incidence of cardiovascular diseases such as hypertension and coronary heart disease, the incidence and mortality of heart failure are also increasing year by year. At present, there are nearly 1 billion people in Europe, and there are at least 15 million patients with heart failure; according to the China Cardiovascular Disease Report 2011, there are currently about 4.2 million patients with heart failure in my country. The prevalence of heart failure in the population is about 1.5% to 2.0%. With age, the incidence is also increasing. The incidence is 2-3% before the age of 75, and the incidence rate o...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63A61K38/22A61P9/04
Inventor 彭红卫赵斌李小鹏雒蓬轶董佳里罗天学徐小萍魏薇钟绍东张宝华
Owner CHENGDU JINKAI BIOTECH CO LTD
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