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Fusion protein and coding gene and application thereof

A technology of fusion protein and coding gene, which is applied in the field of preparation of drugs for treating inflammation and autoimmune diseases, can solve problems such as complex process, low recovery rate, and short half-life, and achieve broad application prospects, short production cycle, large-scale practical effect

Inactive Publication Date: 2011-01-19
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Even after purification and renaturation, some proteins still cannot form correct disulfide bonds and proper space folding, thus affecting their activity
Moreover, the process is complicated and the recovery rate is low, which increases the production cost
[0004] However, biologically active drugs such as cytokines have a short half-life. For example, the half-life of IL-10 in the body is only 2-3 hours. Therapeutic concentrations, however such frequent dosing is inconvenient and painful that some patients cannot tolerate the drug
In the phase II clinical observation, it was also found that the treatment effect is poor when the dosage of IL-10 is small; while increasing the dosage will produce obvious side effects

Method used

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  • Fusion protein and coding gene and application thereof
  • Fusion protein and coding gene and application thereof
  • Fusion protein and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1. Acquisition of a gene encoding a fusion protein with interleukin-10 activity

[0049] Amplify the coding gene of the fusion protein with interleukin-10 activity of the present invention by PCR method, and the specific process comprises the following steps:

[0050] 1. Amplification of human IL-10 gene

[0051] Use TRIzol (purchased from Invitrogen Company, Cat. No. 15596-018) and extract the total RNA of mononuclear cells from isolated healthy human peripheral blood (purchased from Hefei Blood Bank) according to its instructions, and use TRIzol (purchased from Invitrogen Company, Cat. No. 15596-018) , Cat. No. PC108), oligo dT (purchased from Shanghai Sangong, Cat. No. B0181) was used as a primer to synthesize human cDNA. Using the synthesized cDNA as a template, the forward primer 5'-ATGCACAGCTCAGCACTGCTCTG-3' and the reverse primer 5 PCR was carried out under the guidance of '-GTTTCGTATCTTCATTGTCATG-3', and the PCR product was inserted into the TA cloning ...

Embodiment 2

[0062] Example 2, Expression of α-Factor-IL-10-Fc-γ1 in Pichia pastoris and purification of expression products

[0063] 1. Construction of Pichia pastoris expression vector containing α-Factor-IL-10-Fc-γ1

[0064] see figure 1Construct the Pichia pastoris expression vector containing α-Factor-IL-10-Fc-γ1, the specific method is: use restriction endonuclease BamH I (purchased from Promega, product number R6021) and EcoR I (purchased from Promega, product number R6011 ) the PCR product obtained in Example 1 (α-Factor-IL-10-Fc-γ1) was double digested, and then the digested fragment was digested with the same enzyme double digested plasmid pPIC9K with T4 DNA ligase (purchased from Shanghai Sangong, Cat. No. EL0015) were ligated, the ligated products were transformed into E. coli DH5α competent cells, positive recombinants were screened, plasmids were extracted, and sequenced. The sequencing results showed that the plasmid was inserted between the recognition sites of BamHI and E...

Embodiment 3

[0089] Example 3, IL-10 activity detection of fusion protein

[0090] Peripheral blood lymphocyte (PBMC) medium RPMI-1640 (purchased from HyClone, product number SH30809.01B);

[0091] Peripheral blood (purchased from Hefei Blood Bank) was taken from healthy people, and peripheral blood lymphocytes (PBMCs) were separated using Ficoll (purchased from Tianjin Haoyang Biological Products Technology Co., Ltd., product number: LTS1077) according to its instructions. Add 0.1ml human PBMC (2×10 5 cells), were added with different dilution concentrations of the fusion protein (IL-10-IgG / Fc-γ1) purified by S200 molecular sieve chromatography obtained in Example 2 and commercial rhIL-10 (purchased from Rochy Hill Company, article number: 200-10), each dilution set three duplicate wells, at 37 ° C 5% CO 2 After culturing for 24 hours, phytohemagglutinin PHA-P (purchased from sigma, product number: L8754) with a final concentration of 0.1ug / ml was added, and no PHA-P was added to the bl...

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Abstract

The invention discloses a fusion protein and a coding gene and application thereof. The fusion protein provided by the invention is formed by connecting any one of the following proteins at the carboxyl terminal of human interleukin-10 protein: 1) human IgGFc-gamma 1 or mutant protein thereof, and 2) human IgGFc-gamma 2 or mutant protein thereof, wherein the amino acid sequence of the human interleukin-10 protein is an amino acid sequence from 1st to 160th site of the amino terminal of the sequence 1 in a sequence table. Proved by experiments, the IL-10-IgG / Fc fusion protein of interleukin-10activity is produced by using a pichia pastoris fermentation expression system, and a foundation for clinically researching the treatment effect of the IL-10-IgG / Fc fusion protein in the next step.

Description

technical field [0001] The present invention relates to a fusion protein and its encoding gene and application, in particular to a fusion protein with human interleukin 10 activity and its encoding gene, as well as the expression method of the fusion protein and in the preparation of medicines for treating inflammation and autoimmune diseases Applications. Background technique [0002] Interleukin-10 (IL-10), also known as cytokine synthesis inhibitory factor (cytokine synthesis inhibitory factor, CSIF), B cell-derived T cell growth factor (B cell-derived T cell growth factor, B- TCGF) etc. IL-10 is an important cytokine in normal human body. The mature IL-10 has 160 amino acid residues, the monomer molecular weight is 18.7KD, and contains disulfide bonds formed by 4 cysteines (12-108, 62 -114), and its natural active form is a homologous oligodimer with a molecular weight of 38KD linked by non-covalent bonds. IL-10 is a multifunctional cytokine, secreted by a variety of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12N15/81C12P21/02C07K1/20C07K1/16A61K38/20A61K47/48A61K48/00A61P29/00A61P37/02A61K47/68
Inventor 肖卫华郭雨刚康文瑶李光伟
Owner UNIV OF SCI & TECH OF CHINA
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