Fused protein of mutant human interleukin-2 and human serum albumin, and preparation thereof

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

In order to achieve the therapeutic effect, frequent high-dose medication is generally required. Frequent high-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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  • Fused protein of mutant human interleukin-2 and human serum albumin, and preparation thereof
  • Fused protein of mutant human interleukin-2 and human serum albumin, and preparation thereof
  • Fused protein of mutant human interleukin-2 and human serum albumin, and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Cloning of IL2m cDNA

[0028] IL2m cDNA (399bp) was artificially synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., and cloned into the vector PUC57, the insertion site was Sma I, and the recipient bacteria was E. coli DH5α strain.

Embodiment 2

[0029] Example 2: Cloning of HSA cDNA

[0030] HSA cDNA was amplified from the human fetal liver cDNA library by PCR, and the primers used were:

[0031] P1: 5'-AG GTC GAC GATGCACACAAGAGTGAGGTTGCTC-3'

[0032] P2: 5'-GCC AAGCTT TTATAAGCCTAAGGCAGCTTGACTT-3'

[0033] PCR reaction system: 1.5 μl each of 10 μmol / L P1 and P2 primers, 2.5 mmol / L dNTP 4 μl, 10×pfu Buffer 5 μl, 5 U / μl pfu DNA polymerase 0.5 μl, human fetal liver cDNA library 1 μg, 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.

[0034] 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 digested by Sal I and H...

Embodiment 3

[0035] Embodiment 3: Cloning of IL2m cDNA and HSA cDNA fusion gene

[0036] (1) PCR amplification of IL2m cDNA, the primers used are as follows:

[0037] HI1: 5'-CAAGCTGCCTTAGGCTTAGCACCTACTTCAAGTTCTAC-3'

[0038] HI2: 5'-A GCGGCC GCTTAAGTCAGTGTTGAGATGATGC-3'

[0039] PCR reaction system: 1.5 μl each of 10 μmol / L HI1 and HI2 primers, 2.5 mmol / L dNTP 4 μl, 10×pfu Buffer 5 μl, 5 U / μl pfu DNA polymerase 0.5 μl, plasmid template PUC57-IL2m 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.

[0040] (2) PCR amplification of HSA cDNA, the primers used are as follows:

[0041] HI3: 5'-G GAATTC AAAAGAGATGCACACAAGAGTGAGGT-3'

[0042] HI4: 5'-GTAGAACTTGAAGTAGGTGCTAAGCCTAAGGCAGCTTG-3'

[0043] PCR reaction system: 1.5 μl each of 10 μmol / L HI 3 and HI 4 primers, 2.5 mmol / L d...

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Abstract

The invention discloses the preparation method of a fused protein of mutative human interleukin-2(IL-2m) and human serum albumin (HSA),and the product thereof, belonging to the technical field of long-effetive recombinant fused protein drugs. The invention introduces OE-PCR technology to connect IL-2m cDNA and HSA cDNA, without adding any connecting peptide therebetween, obtaining HAS-IL2m cDNA which then expresses in the expression system of a host pichia pastoris; the HAS-IL2m cDNA is integrated with the chromosome of the host. The HAS-IL2m cDNA of the invention comprises a second region with at least 85% of the sequence congenetic with mutative human interleukin-2 and a first region with at least 85% of the sequence congenetic with human serum albumin 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 expression system can be bacteria, barm, insect cell, zooblast, plant cell, etc. The fused protein maintains the physiological characteristics of human interleukin-2 and prolongs the half life of human interleukin-2 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 mutant human interleukin-2 and human serum albumin, which belong to the technical field of long-acting fusion protein drugs. Background technique [0002] Interleukin-2 (Interleukin-2, IL-2) is the core substance of the body's immune response and immune regulation, and has the functions of anti-tumor, anti-virus and enhancing the body's immune ability. It is also a type of hematopoietic factor, and recombinant interleukin-2 is clinically used for thrombocytopenia symptoms caused by chemotherapy. At the same time, he plays a crucial role in regulating the immune activity and strength of T cells. Interleukin-2 was first discovered in peripheral blood lymphocytes by Morgan et al. in 1976. Its molecular weight is 14.5KD. It consists of 133 amino acid residues; it contains a pair of disulfide bonds (C58-C105), but there is also a cysteine ​​at position 125, so there is a pos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/81C12N15/63
Inventor 金坚李华钟金光泽张莲芬李英储敏陈蕴
Owner JIANGNAN UNIV
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