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Method for preparing fusion protein contg. human interferon-alpha 2 and human seralbumin and its products

A human serum albumin, fusion protein technology, applied in the field of long-acting fusion protein drugs, can solve the problems of increasing patient pain, treatment costs, toxic side effects, and high plasma clearance rate

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

IFNα2a and IFNα2b commonly used in clinical practice are easy to be filtered by glomeruli due to their small molecular weight, resulting in high plasma clearance rate and short half-life in vivo (the half-life of subcutaneous administration or intramuscular injection is generally about 8 hours), low bioavailability
In order to achieve the therapeutic effect, frequent high-dose medication is generally required, such as once every other day, each time 8×10 6 IU, while chronic viral hepatitis has a long cycle (generally more than six months), frequent large-dose medication not only increases the patient's pain and treatment costs, but also easily produces toxic and side effects

Method used

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  • Method for preparing fusion protein contg. human interferon-alpha 2 and human seralbumin and its products
  • Method for preparing fusion protein contg. human interferon-alpha 2 and human seralbumin and its products
  • Method for preparing fusion protein contg. human interferon-alpha 2 and human seralbumin and its products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Cloning of IFNα2 cDNA:

[0028] Take normal human peripheral blood plus lymphocyte separation medium, separate human blood leukocytes, and dilute them with DMEM medium containing 5% fetal bovine serum to a cell concentration of 5×10 7 cells / ml, add Newcastle disease virus to 100 hemagglutination units / ml, 37°C, 5% CO 2 , incubate for 1 hour to make the virus adsorb on the cells, centrifuge at 1000×g, discard the supernatant, add fresh DMEM medium with 5% fetal bovine serum, 37°C, 5% CO 2 , and cultured for 18 hours. Total RNA was isolated with an RNA extraction kit, and then reverse-transcribed into cDNA with a one-step RT-PCR kit (purchased from Dalian Bao Biological Co., Ltd.) and IFN α2 was amplified from it. The primers used were as follows:

[0029] Pa2b1: 5'-AGA GTC GAC ATGTGTGATCTGCCTCAA-3'

[0030] Pa2b2: 5'-GCC AAGCTT TCATTCCTTACTTCTTAAACT-3'

[0031] The one-step RT-PCR reaction system was prepared as follows:

[0032] 10×one step R...

Embodiment 2

[0035] Example 2: Cloning of HSA cDNA:

[0036] HSA cDNA was amplified from human fetal liver cDNA library by PCR. The primers used were:

[0037] HSA1: 5'-AGA GTC GAC GATGCACACAAGAGTGAGGTTGCTC-3'

[0038] HSA2: 5'-GCC AAGCTT TTATAAGCCTAAGGCAGCTTGACTT-3'

[0039] The PCR method is as follows: add to the 50 μl reaction system: 3 μl of primers for 10 μmol / L HSA1 and HSA2, 5 μl of 2 mmol / L dNTP, 5 μl of 10×pfu Buffer, 0.5 μl of pfu DNA polymerase at 5 U / μl (dNTP, 10 ×pfu Buffer and pfu DNA polymerase were purchased from Shanghai Bioengineering Technology Service Company, the same below), human fetal liver cDNA library 1 μg, added double distilled water to make up 50 μl. PTC-100 at MJResearch 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.

[0040] The reaction product was analyzed by agarose gel electrophoresis, and a band ...

Embodiment 3

[0041] Example 3: Cloning of fusion fragments of HSA cDNA and IFN α2 cDNA:

[0042] PCR amplification of HSA cDNA: the primers used are as follows:

[0043] Pf1: 5'-CCCTCC GAATTC AAAAGAGATGCACACAAGAGTGAGGTTGCT-3'

[0044] Pf2: 5'-TGGGTTTGAGGCAGATCACATAAGCCTAAGGCAGCTTGAC-3'

[0045] The PCR method is as follows: Add 2.5 μl of 10 μmol / L Pf3 and Pf4 primers to the 50 μl reaction system, 5 μl of 2 mmol / L dNTP, 5 μl of 10×pfu Buffer, 0.5 μl of 5 U / μl pfu DNA polymerase, pBlue- HSA 1ng, add double distilled water to make up 50μl. PTC-100 at MJ Research TM On the PCR instrument, the PCR conditions are: fully denature at 95°C for 5 minutes, anneal at 60°C for 1 minute, extend at 72°C for 4 minutes, denature at 94°C for 1 minute, and cycle 30 times.

[0046] PCR amplification of IFN α2 cDNA: The primers used are as follows:

[0047] Pf3: 5'-GTCAAGCTGCCTTAGGCTTATGTGATCTGCCTCAAACCCA-3'

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

[0049] The PCR method is as f...

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Abstract

The invention relates to a manufacture method and product for confluence albumen of human alpha-2 interferon and human serum albumin that uses superposing PCR technology, connecting IFN alpha-2 cDNA and HAS cDNA, with out any connecting peptide to gain IFN alpha-2 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 alpha 2 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 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) has a secondary structure similar to the corresponding ...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/62C12N15/21C12N15/14C07K19/00C07K14/56C07K14/765C12N1/19
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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