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Fused protein of human serum albumin and human granulocyte colony stimulating factor mutant, and preparation thereof

A technology of human serum albumin and colony-stimulating factor, which can be applied in the directions of hybrid peptides, introduction of foreign genetic material and peptides using vectors, etc., can solve the problems of increasing patient suffering, low actual dosage, and short circulation half-life.

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

Whether it is natural or genetically recombinant G-CSF, the circulating half-life in the human body is very short (t 1 / 2 =1.3~4.2h), the bioavailability is low, in order to achieve the best therapeutic effect, frequent large doses of medication are required, and continuous administration will cause side effects such as drug eruption, fever, myalgia, bone pain, etc., which increases the pain of the patient and gives The clinical application has brought great restrictions, resulting in the actual dosage being far lower than the theoretically required dosage

Method used

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  • Fused protein of human serum albumin and human granulocyte colony stimulating factor mutant, and preparation thereof
  • Fused protein of human serum albumin and human granulocyte colony stimulating factor mutant, and preparation thereof
  • Fused protein of human serum albumin and human granulocyte colony stimulating factor mutant, and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: hGCSF m cDNA cloning

[0036] hGCSF was artificially synthesized by Shanghai Sangon Biotechnology Service Co., Ltd. m The cDNA (541bp) was cloned into the vector pUC57, the insertion site was EcoR V, and the recipient bacterium was Escherichia coli DH5α strain.

Embodiment 2

[0037] Example 2: Cloning of HSA cDNA

[0038] HSA cDNA was amplified from the human fetal liver cDNA library by PCR, and the primers used were:

[0039] PH1: 5'-CAGC GAATTC GATGCACACAAGAGTGAGGTTGCTC-3'

[0040] PH2: 5'-CACC GCGGCCGCT TTATAAGCCTAAGGCAGCTTGACTT-3'

[0041] The underlined part of PH1 is the EcoR I restriction site, and the underlined part of PH2 is the NotI restriction site

[0042] 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 min; denaturation at 94°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 3 min, 30 cycles; extension at 72°C for 10 min.

[0043] 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...

Embodiment 3

[0044] Example 3: Containing HSA cDNA and hGCSF m Construction of Cloning Vector of cDNA Fusion Gene

[0045] (1) The constructed pBlu2KSP-HSA vector contains Saul I and Not I restriction sites at the C-terminus of the HSA gene, which can be used in hGCSF m The upstream and downstream primers of the gene were respectively introduced with Saul I and Not I restriction sites, designed to make hGCSF m After the gene was digested by Saul I and Not I, it was ligated in frame with pBlu2KSP-HSA which was digested by Saul I and Not I.

[0046] (2) In-frame digestion and ligation of HSA cDNA and hGCSF m cDNA fusion gene

[0047] Digest pUC57-hGCSF with Saul I and Not I m and pBlu2KSP-HSA vector, use the PCR Fragment Gel Recovery Kit to recover hGCSF m gene and pBlu2KSP-HSA vector. The two recovered fragments were ligated with T4 DNA ligase and transformed into Escherichia coli DH5α. The transformant was spread on an LB agar plate containing X-gal, IPTG and 100 μg / mL ampicillin, a...

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Abstract

The invention relates to a preparation method of a fused protein of human serum albumin and mutant human granulocyte colony stimulating factor, and the product thereof, belonging to the technical field of long-effetive recombinant fused protein drugs. Human serum albumin(HSA) cDNA and mutant human granulocyte colony stimulating factor(hGCSF) cDNA are directly connected, without adding any connecting peptide therevetween, obtaining HAS- hGCSFm cDNA which is then integrated with a cell of the host to express. The fused protein of the invention comprises a first region with at least 85% of the sequence congenetic with human serum albumin and a second region with at least 85% of the sequence congenetic with the mutant human granulocyte colony stimulating factor, and the two regions are directly connected without any connecting peptide therebetween. 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 host cell can be bacteria, barm, insect cell, zooblast, plant cell, etc. The fused protein maintains the physiological characteristics of the mutant human granulocyte colony stimulating factor and prolonges the half life of the mutant human granulocyte colony stimulating factor within human body; therefore the fused protein is of good application prospect in pharmacy.

Description

technical field [0001] The invention relates to a preparation method and product of a fusion protein of human serum albumin and human granulocyte colony-stimulating factor mutant, belonging to the technical field of long-acting fusion protein drugs. Background technique [0002] Human serum albumin (Human serum albumin, HSA) is the main protein component in plasma, and its concentration in plasma is 40 mg / mL (Phillip P. Minghettis et al., THE JOURNAL OF BIOLOGICAL CHEMISTRY, 1986 261: 6747-6757) , in addition to its function of maintaining plasma osmotic pressure, it can also bind endogenous and / or exogenous ligands, including hormones, toxic metabolites, drugs, etc. By binding these ligands, HSA can regulate hormone activity, toxicity of endogenous and / or exogenous substances, and availability of drugs (Ji-Sook Ha et al., Biochimica et Biophysica Acta, 20031640: 119-128). The Half HSA constructed by David S. Park et al. (truncating the first 297 amino acid residues of HSA)...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/81C12N15/63
Inventor 金坚朱书峰张莲芬李英储敏陈蕴
Owner JIANGNAN UNIV
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