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Fusion protein in soluble receptor II - antibody Fc section of human tumor necrosis factor

A tumor necrosis factor and fusion protein technology, applied in the field of human tumor necrosis factor soluble receptor II-antibody Fc segment fusion protein, can solve the problems of large side effects and insignificant effects of drugs, and achieve the goal of improving expression level and reducing screening intensity. Effect

Inactive Publication Date: 2007-07-25
TAIDA RES CENT OF LIFE SCI & TECH INST OF HEMATOLOGY CHINES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Current rheumatoid arthritis drug treatment includes hormones and immunosuppressants, etc., the effect is not obvious, and the side effects of drugs are relatively large

Method used

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  • Fusion protein in soluble receptor II - antibody Fc section of human tumor necrosis factor
  • Fusion protein in soluble receptor II - antibody Fc section of human tumor necrosis factor

Examples

Experimental program
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Effect test

Embodiment 1

[0015] Embodiment 1 PCR amplification gene: with SEQ ID No.4 (5'-AAAAAA GTTTAAAC ATGGTTCGACCATAGAACT-3') is the forward primer, with SEQ ID No.5 (5'AAAAAA GCTAGC TTAGTCTTTTTCCCTCGTAGACT-3') was used as the reverse primer, and the CHO cell cDNA library (Clontech Company) was used as the template. The PCR reaction was carried out in a total volume of 50 μL, and the reaction conditions were denaturation at 94°C for 5 minutes, followed by cycling at 94°C for 50 seconds. , annealing at 58°C for 1.5 minutes, extension at 72°C for 1 minute, a total of 30 cycles, and then extending at 72°C for 10 minutes, amplified a DNA fragment SEQ ID No.3 consistent with the expected size (564bp).

Embodiment 2

[0016] Example 2 Construction of pT-dhfr expression vector: The gel-recovered PCR product and the vector pTandem-1 (Novagen) were double-digested with PmeI and BlpI, respectively, and the reagents were recovered from 1% agarose gel by gel The box was recovered, then ligated (16°C, 16h), transformed into Escherichia coli DH-5α competent cells, positive clones were selected, plasmids were extracted and verified by double enzyme digestion analysis, and DNA sequencing was carried out for identification. The sequencing results were consistent with SEQ ID No. 3. The sequences are identical, and the constructed expression plasmid is called pT-dhfr. The construction strategy is shown in FIG. 3 .

Embodiment 3

[0017] Example 3 Two primers of SEQ ID No.8 and SEQ ID No.9 were synthesized according to SEQ ID No.6, and the human lung cDNA library (Clontech Company) was used as a template for PCR reaction to amplify human tumor necrosis factor soluble receptor II gene sequence (TNFRII), the PCR product is TNFRII-pcrp.

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Abstract

This invention discloses fusion protein of soluble receptor II of human tumor necrosis factor and Fc fragment of antibody. The amino acid sequence of the fusion protein is shown in SEQ ID No.2. This invention utilizes IRES sequence of double-cistronic vector pTandem-1 to ligate the gene sequence of the fusion protein with DHFR gene, which is used as a screening marker. This invention can reduce the screening intensity and raise the expression level. This invention can effectively and stably produce fusion protein of soluble receptor II of human tumor necrosis factor and Fc fragment of antibody.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a human tumor necrosis factor soluble receptor II-antibody Fc segment fusion protein. Background technique [0002] The successful research and development of recombinant soluble receptor drugs is a major breakthrough in the development of biotechnology drugs in recent years. The diseases treated include cancer, cardiovascular diseases and autoimmune diseases. In the early 1980s, in patients with rheumatoid arthritis, it was found that the content of tumor necrosis factor (tumor necrosis factor-α, TNF) in serum was much higher than that of normal people. In the later animal model of mouse rheumatoid arthritis, it also showed that the level of serum tumor necrosis factor was significantly increased. The extracted tumor necrosis factor soluble receptor-II (TNFR-II) is used to neutralize excessive TNF in the body to treat mice with rheumatoid arthritis, and the curative...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62
Inventor 徐斌张翔宇文峰杨萍高祁韩忠朝
Owner TAIDA RES CENT OF LIFE SCI & TECH INST OF HEMATOLOGY CHINES
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