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Fused protein of human brain natriuretic peptide diad [(BNP)2] and human serum albumin (HAS), and preparation thereof

A human serum albumin, fusion protein technology, applied in the field of long-acting fusion protein drugs, can solve the problems of frequent injection, high treatment cost, poor stability, etc.

Inactive Publication Date: 2008-10-08
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Like other recombinant peptide / protein drugs that have been widely used clinically, recombinant BNP drugs have low solubility, poor stability, and a half-life in vivo of only 22 minutes, so they must be administered in high doses continuously to be effective
This type of drug has high treatment costs, low drug utilization, and frequent injections by patients, resulting in the actual dosage being far lower than the required amount

Method used

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  • Fused protein of human brain natriuretic peptide diad [(BNP)2] and human serum albumin (HAS), and preparation thereof
  • Fused protein of human brain natriuretic peptide diad [(BNP)2] and human serum albumin (HAS), and preparation thereof
  • Fused protein of human brain natriuretic peptide diad [(BNP)2] and human serum albumin (HAS), and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: (BNP) 2 cDNA cloning

[0029] Artificially synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. (BNP) 2 The cDNA (195bp) was cloned into the vector pBlu2KSP, the insertion site was Sma I, and the recipient bacterium was Escherichia coli JM109 strain.

Embodiment 2

[0030] Example 2: Cloning of HSA cDNA

[0031] HSA cDNA was amplified from the human fetal liver cDNA library by PCR, and the primers used were:

[0032] PH1: 5'-AG GTC GAC GATGCACACAAGAGTGAGGTTGCTC-3'

[0033] PH2: 5'-GCC AAGCTT TTATAAGCCTAAGGCAGCTTGACTT-3'

[0034] PCR reaction system: 1.5 μl of 10 μmol / L PH1 and PH2 primers, 4 μl of 2.5 mmol / L dNTP, 5 μl of 10×pfu Buffer, 0.5 μl of 5 U / μl pfu DNA polymerase, 1 μg of human fetal liver cDNA library, plus double Make up 50 μl with distilled water. PCR reaction program: pre-denaturation at 95°C for 5 minutes; denaturation at 94°C for 1 minute, annealing at 60°C for 1 minute, extension at 72°C for 3 minutes, 30 cycles; extension at 72°C for 10 minutes.

[0035] The reaction product was analyzed by agarose gel electrophoresis, and the target band appeared in the loading lane, and the 1.8kb target fragment was purified with the PCR Fragment Gel Recovery Kit. The purified target fragment and the vector pBlu2KSP were digeste...

Embodiment 3

[0036] Example 3: (BNP) 2 Cloning of cDNA and HSA cDNA fusion gene

[0037] (1) (BNP) 2 For PCR amplification of cDNA, the primers used are as follows:

[0038] PB1: 5'-GCGCGC GAATTC AAAAGATCTCCAAAGATGGTCCAAGG-3'

[0039] PB2: 5'-ACCTCACTCTTGTGTGCATCGTGACGTCTCAGGACCTTGC-3'

[0040] PCR reaction system: 1.5 μl of 10 μmol / L PB1 and PB2 primers, 4 μl of 2.5 mmol / L dNTP, 5 μl of 10×pfu Buffer, 0.5 μl of 5 U / μl pfu DNA polymerase, plasmid template pBlu2KSP-(BNP) 2 1ng, add double distilled water to make up 50μl. PCR reaction program: pre-denaturation at 95°C for 5 minutes; denaturation at 94°C for 1 minute, annealing at 65°C for 1 minute, extension at 72°C for 30 seconds, 30 cycles; extension at 72°C for 10 minutes.

[0041] (2) PCR amplification of HSA cDNA, the primers used are as follows:

[0042] PH3: 5'-GCAAGGTCCTGAGACGTCACGATGCACACAAGAGTGAGGT-3'

[0043] PH4: 5'-CATAAG GCGGCCGC TTATTATAAGCCTAAGGCAGCTTG-3'

[0044] PCR reaction system: 1.5 μl each of 10 μmol / L PH 3...

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Abstract

The invention discloses a fused protein from human brain natriuretic peptide(BNP)2 and human serum albumin (HSA) and the preparation method thereof, belonging to the technical field of long-effetive recombinant fused protein drugs. The invention introduces OE-PCR technology to splice two BNP cDNAs(called (BNP)2 for short) and HAS cDNA, without adding any connecting peptide therebetween, obtaining fused gene(BNP)2-HSA cDNA which is then integrated with the chromosome of the host and expresses in the expression system of the host. The fused protein of the invention comprises a first region with at least 85% of the sequence congenetic with human brain natriuretic peptide and a second region with at least 85%of the sequence congenetic with human serum albumin. Under the premise of not being changed in characteristics, the fused protein is capable of the substitution, deletion or addition of specific amino acid residues. The fused protein maintains the physiological characteristics and improves the solubility of human brain natriuretic peptide and prolongs the half life of human brain natriuretic peptide within human body; therefore the fused protein is of good application prospect in pharmacy.

Description

technical field [0001] Human brain natriuretic peptide duplex [(BNP) 2 ] and human serum albumin (HSA) fusion protein preparation method and product, belonging to the technical field of long-acting fusion protein drugs. Background technique [0002] Brain Natriuretic Peptide (BNP) is a member of the natriuretic peptide family discovered by Sudoh et al. in pig brain in 1988. It is mainly secreted and synthesized by the ventricle. The precursor contains 108 amino acids, and the processed release contains 32 amino acids. amino acids of the mature BNP molecule. The main characteristic of BNP is to antagonize the action of the renin-angiotensin-aldosterone system, that is, by inhibiting the secretion of renin and aldosterone, increasing the glomerular filtration rate and inhibiting the sodium reabsorption of the renal medullary collecting duct to promote sodium excretion and diuresis . It can also dilate blood vessels by directly relaxing vascular smooth muscle and antagonizin...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/81C12N15/63
Inventor 金坚丁月娣张莲芬李英储敏陈蕴
Owner JIANGNAN UNIV
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