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Method for preparing fusion protein contg. human interferon-beta and human seralbumin and its products

A technology of human serum albumin and fusion protein, which is applied in the field of long-acting fusion protein drugs and can solve the problems of reducing the curative effect of fusion proteins

Inactive Publication Date: 2006-09-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the existence of allotypes of human IgG, corresponding antibodies may be produced in the body of the drug user and reduce the efficacy of the fusion protein

Method used

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  • Method for preparing fusion protein contg. human interferon-beta and human seralbumin and its products
  • Method for preparing fusion protein contg. human interferon-beta and human seralbumin and its products
  • Method for preparing fusion protein contg. human interferon-beta and human seralbumin and its products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Cloning of IFNβ cDNA

[0025] Whole blood genomic DNA purification system (purchased from Shanghai Bioengineering Technology Service Company) was used to extract genomic DNA from healthy human blood concentrated leukocytes according to the operating instructions. The extracted DNA was treated with restriction endonuclease EcoRI or HindIII, and the cDNA of IFNβ was amplified by PCR, and the primers used were as follows:

[0026] Pb1: 5'-TA GTC GAC ATGAGCTACAACTTGCTTGG-3'

[0027] Pb2: 5'-AG AAGCTT TCAGTTTCGGAGGTAACCTG-3'

[0028] The PCR method is as follows:

[0029] Add to the 50μl reaction system: 3μl of 10μmol / L Pb1 and Pb2 primers, 5μl of 2mmol / L dNTP, 1μl of 10×pfu Buffer, 0.5μl of 5U / μl pfu DNA polymerase (dNTP, 10×pfuBuffer and pfu All DNA polymerases were purchased from Shanghai Bioengineering Technology Service Company, the same below), and 1 μg of human genomic DNA treated with the enzyme was added to 50 μl of double distilled water. PTC-100...

Embodiment 2

[0031] Example 2: Cloning of HSAcDNA

[0032] HSA cDNA was amplified from human fetal liver cDNA library by PCR. The primers used were:

[0033] HSA1: 5'-AGA GTC GAC GATGCACACAAGAGTGAGGTTGCTC-3'

[0034] HSA2: 5'-GCC AAGCTT TTATAAGCCTAAGGCAGCTTGACTT-3'

[0035] The PCR method is as follows:

[0036] Add to the 50 μl reaction system: 3 μl of 10 μmol / L HSA1 and HSA2 primers, 5 μl of 2 mmol / L dNTP, 1 μl of 10×pfu Buffer, 0.5 μl of 5 U / μl pfu DNA polymerase, 1 μg of human fetal liver cDNA library, Add double distilled water to make up 50 μl. PTC-100 at MJ Research TM On the PCR instrument, the PCR conditions are: fully denature at 95°C for 5 minutes, denature at 94°C for 1 minute, anneal at 60°C for 1 minute, extend at 72°C for 90 seconds, and cycle 30 times.

[0037] The reaction product was analyzed by agarose gel electrophoresis, and a band of expected size (about 1.8 kb) appeared in the loading lane. A 1.8 kb target fragment was purified with a PCR Fragment Gel Reco...

Embodiment 3

[0038] Embodiment 3: Cloning of fusion gene of IFNβ cDNA and HSA cDNA

[0039] PCR amplification of IFNβ cDNA:

[0040] The primers used are as follows:

[0041] Pf1: 5'-CCCTCC GAATTC AAAAGAATGAGCTACAACTTGCTTGGA-3'

[0042] Pf2: 5'-ACCTCACTCTTGTGTGCATCGTTTCGGAGGTAACCTGTAA-3'

[0043] The PCR method is as follows:

[0044] Add to the 50μl reaction system: 10μmol / L Pf1 and Pf2 primers 2.5μl each, 2mmol / L dNTP 5μl, 10×pfu Buffer 1μl, 5U / μl pfu DNA polymerase 0.5μl, pBlue-IFNβ 1ng, add Make up 50 μl of double distilled water. PTC-100 at MJ Research TM On the PCR instrument, the PCR conditions are: fully denature at 95°C for 5 minutes, anneal at 65°C for 1 minute, extend at 72°C for 90 seconds, denature at 94°C for 1 minute, and cycle 30 times.

[0045] PCR amplification of HSA cDNA:

[0046] The primers used are as follows:

[0047] Pf3 5'-TTACAGGTTACCTCCGAAACGATGCACACAAGAGTGAGGTT-3'

[0048] Pf4: 5'-CATAAG GCGGCCGC TTATTATAAGCCTAAGGCAGCTTGA-3'

[0049] The PCR metho...

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PUM

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Abstract

The invention relates to a manufacture method and product for confluence albumen of human beta interferon and human serum albumin that uses superposing PCR technology, connecting IFN beta cDNA and HAS cDNA, with out any connecting peptide to gain IFN beta-HSA cDNA melting gene to be integrated into host chromosome to take expression. The confluence albumen includes the first area of at least 85% sequence same source as human beta-interferon and the second area of at least 85% sequence of same source of human serum albumin. The invention has good application prospect in medicine field.

Description

technical field [0001] The preparation method and product of the fusion protein of human beta interferon and human serum albumin belong to the technical field of long-acting fusion protein medicine. Background technique [0002] Human serum albumin (Human serum albumin, HSA) is the main protein component in plasma, and the concentration in plasma is 40mg / ml (Phillip P.Minghettis et al., THE JOURNAL OFBIOLOGICAL 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, 2003 1640: 119-128) . HalfHSA (truncating the first 297 amino acid residues of HSA) constructed by David S. Park et al. has a secondary structure similar to the corresponding re...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/62C12N15/22C12N15/14C07K19/00C07K14/565C07K14/765C12N1/19
Inventor 金坚雷楗勇杨健良张莲芬窦文芳徐钰李洁
Owner JIANGNAN UNIV
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