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TNFr and stability and anti-aggregation performance enhanced Fc fragment fusion protein as well as preparation method and application thereof

A fusion protein and anti-aggregation technology, applied in the field of Fc fragment fusion protein and its preparation, can solve the problems of poor stability and weak anti-aggregation ability, and achieve the effects of good stability, good anti-aggregation ability and reducing the risk of clinical use

Inactive Publication Date: 2018-04-20
武汉班科生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to overcome the defects of poor stability and weak anti-aggregation ability of Fc fusion proteins in the prior art, provide a kind of Fc fragment fusion protein with enhanced stability and anti-aggregation of TNFr and its preparation method and application, which is different from the existing Compared with TNFr-Fc fusion protein, it has better stability and more anti-aggregation characteristics, which helps to improve the efficacy of TNFr-Fc and reduce side effects caused by instability or easy aggregation

Method used

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  • TNFr and stability and anti-aggregation performance enhanced Fc fragment fusion protein as well as preparation method and application thereof
  • TNFr and stability and anti-aggregation performance enhanced Fc fragment fusion protein as well as preparation method and application thereof
  • TNFr and stability and anti-aggregation performance enhanced Fc fragment fusion protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Amplification of TNFr-Fc and TNFr-S23 fragment genes

[0024] A human blood sample was taken, and the total cell RNA was extracted with Trizol Reagent (Invitrogen), and the obtained total cell RNA was dissolved in 50 μL of RNase-free water. Then use the M-MLV Reverse Transcriptase Kit (Promega Company), using random primers, and reverse transcription to obtain cDNA.

[0025] According to the Fc gene sequence of IgG (GenBank: KJ905798.1), analyze its amino acid sequence and design the forward and reverse primers of the Fc gene to amplify the target fragment:

[0026] Forward primer

[0027] 5-GAG CCC AAA TCT AGC GAC AAAACT CAC AC-3(1)

[0028] 5-ACGC GGCCCAGCCGGCC TTGCCCGCCCAGGTGGCATTTAC-3(2) (the horizontal line is marked as Sfi I restriction site);

[0029] Reverse primer

[0030] 5-GCCCTC CTCGAG TCATTTACCCGGAGACAGGGAG-3(1) (the horizontal line is the Xho I restriction site)

[0031] 5-GTCGCCAGTGCTCCCTTCAGCTGGG-3(2)

[0032] Use forward primers (1) / (2) and reverse pri...

Embodiment 2

[0045] Example 2: Expression and purification of TNFr-Fc and TNFr-S23

[0046] PEI (2mg / mL PEI) (Polyethylenimine "Max", (Mw 40,000)-High Potency Linear PEI, Polysciences, Inc., Catalog No.: 24765-2, 2g) was used for each 40ug of TNFr-Fc and TNFr-S23 plasmids. 293F cells were transfected and cultured with 40ml 293F expression medium (invitrogen) at 37°C and 150rpm shaker for 6 days.

[0047] Subsequently, the supernatant of 293F cells was collected by centrifugation (6000g, 4°C, 15min) (the supernatant uses mouse anti-human IgG Fc as the primary antibody and HRP-labeled goat anti-mouse IgG as the secondary antibody for Western blot detection. The results are as follows: figure 1 As shown, lane 1: Marker; lane 2: TNFr-Fc cell supernatant; lane 3: TNFr-S23 cell supernatant). After the collected supernatant is filtered, TNFr-Fc, TNFr-S23 protein. Subsequently, the target protein was concentrated by ultrafiltration with an ultrafiltration centrifuge tube (Merck Millipore) with a molec...

Embodiment 3

[0048] Example 3: Molecular conformation and existence of TNFr-Fc and TNFr-S23 (monomer, dimer, etc.)

[0049] AKTA analysis of TNFr-Fc and TNFr-S23 existence form: Concentrate the purified TNFr-Fc and TNFr-S23 protein to 1mg / ml, PBS (pH7.4) as the elution buffer, and pass Column Superdex 200 Increase 10 / 300GL , Where the flow rate is 0.5ml / min, and its existence form is detected. At the same time, 1mg / ml Etanercept and Yisaipu protein were also passed through Column Superdex 200 Increase 10 / 300GL molecular sieve to compare the existing forms of the protein.

[0050] The result is image 3 As shown, Yisaipu (A), Etanercept (B), TNFr-Fc (C), TNFr-S23 (D), compared with the standard curve can be found, TNFr-Fc (C), TNFr-S23 (D) protein molecular weight About 140kDa, the results show that they exist in the form of dimers. Compared with the molecular sieve diagrams of Etanercept and Yisaipu protein, the peak of TNFr-S23 is more single, indicating that the protein is more stable and si...

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Abstract

The invention discloses TNFr and stability and anti-aggregation performance enhanced Fc fragment fusion protein as well as a preparation method and application thereof. A pair of disulfide bonds are respectively introduced into CH2 and CH3 structure domains of the Fc fragments of the TNFr-Fc fusion protein; the mutation type TNFr-S23 fusion protein is obtained. Compared with wild type TNFr-Fc, theprotein has the advantages that the stability is higher; the production, purification and storage cost of the protein can be reduced. Compared with wild type TNFr-Fc, the protein has the advantages that the anti-aggregation performance is better; the clinic use risk caused by protein aggregation can be reduced.

Description

Technical field [0001] The invention relates to the field of biotechnology, in particular to a fusion protein of TNFr and an Fc fragment with enhanced stability and anti-aggregation properties and a preparation method thereof. Background technique [0002] Human tumor necrosis factor (TNF-α) is a cytokine produced by activated monocytes / macrophages and has a variety of biological effects. TNF-α can induce tumor cell necrosis or apoptosis, and at the same time is the main cytokine that mediates inflammation. Recombinant human type II tumor necrosis factor receptor-Fc fusion protein (TNFr-Fc) is a protein drug approved by the US FDA for the treatment of autoimmune diseases such as rheumatoid arthritis. Etanercept (Enbrel TM) is a commercially available TNFr-Fc fusion protein and one of the most effective anti-TNF drugs. It is also a very high-selling product in biotechnology drugs. The TNFr-Fc fusion protein produced by China CITIC Guojian has been produced and marketed under the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85A61K38/17A61K47/68A61P37/02
CPCA61K38/00C07K14/7151C07K2319/30C12N15/85
Inventor 龚睿
Owner 武汉班科生物技术有限公司
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