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TNF (Tumor Necrosis Factor)-related apoptosis-inducing ligand fusion protein and preparation method thereof

A technology of apoptosis-inducing ligand and tumor necrosis factor, which is applied in the field of tumor necrosis factor-related apoptosis-inducing ligand fusion protein and its preparation, to reduce costs, enhance anti-tumor effects, and inhibit angiogenesis

Active Publication Date: 2014-07-23
英百瑞(杭州)生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the strong hydrophobicity of TRAIL and NC1, the traditional method encountered great difficulties in refolding their fusion protein inclusion bodies. Therefore, when the fusion protein drug is developed in industry, it is hoped that relevant technical personnel can provide Simple, effective and low-cost renaturation method

Method used

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  • TNF (Tumor Necrosis Factor)-related apoptosis-inducing ligand fusion protein and preparation method thereof
  • TNF (Tumor Necrosis Factor)-related apoptosis-inducing ligand fusion protein and preparation method thereof
  • TNF (Tumor Necrosis Factor)-related apoptosis-inducing ligand fusion protein and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Amplification of TRAIL114-281 and NC1 fragments

[0031] (1) Amplification of TRAIL114-281

[0032] Extract human prostate total mRNA: Grind frozen human prostate tissue small pieces (about 100mg) into powder with liquid nitrogen, add 1ml TRIZOL reagent and continue grinding repeatedly, transfer to 1.5ml RNase-free EP tube, place at room temperature for 5min, add 0.2 Vigorously shake in ml chloroform for 15 seconds, and place at room temperature for 3 minutes. Centrifuge at 12000g at 4°C for 15 minutes, and transfer the upper aqueous phase to another RNase-free EP tube. Add 0.5ml of isopropanol and let stand at room temperature for 10min to precipitate RNA. Centrifuge at 12,000 g at 4°C for 10 min to remove the supernatant, and wash the precipitate with 75% ethanol. Centrifuge at 12,000 g at 4°C for 5 minutes to remove the supernatant, dissolve in 50 μl DEPC water after drying, and store at -70°C. Using oligodT as primer, human prostate total RNA as temp...

Embodiment 2

[0035] Example 2: Construction of TRAIL-(G)n-NC1 and TRAIL-(G)n-TD fusion protein expression vectors and their prokaryotic expression

[0036] ⑴ Synthesis protein expression vector construction:

[0037] The pET28a vector contains NcoI, BamHI and XhoI. TRAIL114-281 was digested with NcoI and BamHI and ligated with pET28a after the same digestion to construct pET28a-TRAIL114-281. The pET28a-TRAIL114-281 fragment was ligated to pET28a-TRAIL114-281 after NC1 was digested with BamHI and XhoI to construct a pET28a-TRAIL(114-281)-linker-NC1 expression vector. see image 3 ： (a): Monomer composition of TRAIL-(G)n-NC1 fusion protein: TRAIL114-281, peptide linker and NC1 domain. The NC1 domain is composed of three parts: trimerization domain (TD), hinge region and endostatin (endostatin); (b): schematic diagram of TRAIL-(G)n-NC1 fusion protein trimer; (c): TRAIL- (G) Ideal three-dimensional structure diagram of n-NC1 trimer; (d): Ideal schematic diagram of TRAIL-(G)n-NC1 exerting...

Embodiment 3

[0040] Example 3: Acquisition and preliminary purification of TRAIL-(G)n-NC1 and TRAIL-(G)n-TD fusion protein inclusion bodies

[0041]Resuspend the cells with 50mM PBS (pH 7.4), 20mM EDTA, 0.5-3% (v / v) Triton X-100, vortex for 5min, crush BL21 mechanically with French pressure, centrifuge to remove the supernatant, and obtain crude inclusion bodies. Wash with 50mM PBS (pH 7.4), 1M NaCl, 1-6 M Urea, 0.5-3% (v / v) Triton X-100, and centrifuge. repeat three times. Wash with sterile water three times. Wash again with 50mM PBS (pH 7.4), centrifuge to remove the supernatant, and the obtained fusion protein inclusion body is initially purified. Dissolve inclusion bodies with 8-10M Urea or 5-8M GdmCl+50mM NaH2PO4+30-300 mM DTT (pH 8-10), let stand at room temperature for 5 hours, centrifuge to take the supernatant, which is the dissolved inclusion bodies.

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Abstract

The invention provides a fusion protein of the extracellular part of the tumor necrosis factor-related apoptosis-inducing ligand and the N1 domain or the trimerization domain of type XVIII collagen, and the peptide linker is [GlyGlyGlyGlySer]n, where n is an integer of 0-4. By constructing the recombinant fusion protein gene; expressing the fusion protein gene and performing renaturation. Among them, the renatured fusion protein exhibits strong activity of inducing tumor cell apoptosis. The present invention utilizes the design of the fusion gene of type XVIII collagen NC1 domain fused with TRAIL, which can prolong the half-life of the corresponding fusion protein in experimental animals and overcome the shortcoming that the half-life of TRAIL is only a few minutes; NC1 or trimerization domain has extremely strong trimerization properties , after fusion with TRAIL, its trimer can be stabilized, thereby maintaining the activity of inducing tumor cell apoptosis; the endostatin domain contained in the NC1 domain has the effect of inhibiting angiogenesis, and this effect can cooperate with the apoptosis effect of TRAIL to enhance fusion Antitumor effects of proteins. The method of the invention can effectively reduce the cost of laboratory or industrial production of fusion protein.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, relates to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and type XVIII collagen NC1 domain or trimerization domain (trimerization domain, TD) synergistic anti-tumor effect and protein stability ) fusion protein, and the renaturation and purification method of fusion protein inclusion body. technical background [0002] Tumor necrosis factor (TNF) related apoptosis-inducing ligand (TNF related apoptosis-inducing ligand, TRAIL) is the third apoptotic factor of the TNF family discovered after TNF and FasL. biologic drugs. TRAIL was cloned from the myocardial cDNA library by Wiley et al. It was named because its amino acid sequence has the structural characteristics of the TNF superfamily and can induce the apoptosis of Jurkat cells and Epstein-Barr virus-transformed human lymphocytes. Many preclinical studies have shown that TRAIL can effectively induce apoptosis in v...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/70C07K1/16
Inventor 潘利强陈枢青
Owner 英百瑞(杭州)生物医药有限公司
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