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Fusion protein of human vasonatrin peptide and human serum albumin and preparation thereof

A technology of human vascular natriuretic peptide and human serum albumin, which can be applied to peptides, hybrid peptides, specific peptides, etc., can solve the problems of low solubility of recombinant VNP drugs, low drug availability, and low actual dosage, and achieve good application prospects. , Conducive to purification, rapid growth effect

Inactive Publication Date: 2014-12-24
SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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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 in clinical practice, recombinant VNP drugs have low solubility, poor stability, and a half-life in vivo of only 3 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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  • Fusion protein of human vasonatrin peptide and human serum albumin and preparation thereof
  • Fusion protein of human vasonatrin peptide and human serum albumin and preparation thereof
  • Fusion protein of human vasonatrin peptide and human serum albumin and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Cloning of hVNP cDNA

[0038] The hVNP cDNA (195bp) was artificially synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., and cloned into the vector pBlu2KSP, the insertion site was Sma I, and the recipient bacterium was Escherichia coli DH5a strain.

Embodiment 2

[0039] Example 2: Cloning of HSA cDNA

[0040] HSA cDNA was amplified from the human fetal liver cDNA library by PCR, and the primers used were:

[0041] PH1: 5'-AG GTC GAC GATGCACACAAGAGTGAGGTTGCTC-3'

[0042] PH2: 5'-GCC AAGCTT TTATAAGCCTAAGGCAGCTTGACTT-3'

[0043] 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.

[0044] 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 carrier pBlu2KSP were digest...

Embodiment 3

[0045] Embodiment 3: Cloning of hVNP cDNA and HSA cDNA fusion gene

[0046] (1) PCR amplification of hVNP cDNA, the primers used are as follows:

[0047] PB1: 5'-CAAGTGTCAACTCCAACTCTTGTAG-3'

[0048] PB2: 5'-GTATCTAAAAAGAGTTACAACCCAAACC-3'

[0049] PCR reaction system: 1.5 μl each of 10 μmol / L PB1 and PB2 primers, 2.5 mmol / L dNTP 4 μl, 10×pfu Buffer 5 μl, 5 U / μl pfu DNA polymerase 0.5 μl, plasmid template pBlu2KSP-hVNP 1ng, add 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 65°C for 1 minute, extension at 72°C for 30 seconds, 30 cycles; extension at 72°C for 10 minutes.

[0050] (2) PCR amplification of HSA cDNA, the primers used are as follows:

[0051] PH3: 5'-GCAAGGTCCTGAGACGTCACGATGCACACAAGAGTGAGGT-3'

[0052] PH4: 5'-CATAAG GCGGCCGC TTATTATAAGCCTAAGGCAGCTTG-3'

[0053] PCR reaction system: 1.5 μl each of 10 μmol / L PH3 and PH4 primers, 2.5 mmol / L dNTP 4 μl, 10×...

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Abstract

The invention relates to fusion protein of human vasonatrin peptide and human serum albumin and a preparation method thereof and belongs to the technical field of long-acting recombinant fusion protein medicines. According to the invention, an overlapping PCR (polymerase chain reaction) technology is used, hVNP and HSA cDNA are spliced, no linker peptide is added in the middle, an obtained hVNP-HSA cDNA fusion gene is integrated into the chromosome of a parasitifer, and is expressed in the expression system of the parasitifer. The fusion protein provided by the invention comprises a first region which is homologous to at least 85 percent of sequence of the human vasonatrin peptide and a second region which is homologous to at least 85 percent of the sequence of the human serum albumin. The replacement, the deletion or the addition of an individual amino acid residue can be performed under the premise of not changing the characteristics of the fusion protein. The fusion protein provided by the invention has the advantages as follows: the half life period inside a body can be prolonged on the basis of keeping the physiological characteristics of the human vasonatrin peptide, the solvability is improved, and the fusion protein has an excellent application prospect in the field of pharmacology.

Description

technical field [0001] The invention belongs to the technical field of long-acting fusion protein drugs, and relates to a fusion protein of human vascular natriuretic peptide (hVNP) and human serum albumin (HSA) and a preparation method thereof. Background technique [0002] The high morbidity and high mortality of cardiovascular and cerebrovascular diseases have become a worldwide consensus, and heart failure (HF) is a serious stage of heart disease in most patients with organic heart disease. Recombinant B-type natriuretic peptide (BNP) has become a routine drug for the treatment of HF, but BNP is an effective vasodilator, its diuretic effect is still questionable, and at high doses, it is prone to Symptoms of hypotension. Vasonatrin peptide (VNP) is based on the in-depth study of the relationship between the structure and biological activity of each member of the human natriuretic peptide family by Wei et al., adding the C-terminus of ANP to the C-terminus of the human C...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/81
Inventor 张雁云金坚钱柳金敏王慧姜曰水崔燕生陈蕴刘强
Owner SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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