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Preparation method of protein 4-1BBL

A protein and recombinant protein technology, applied in the field of 4-1BBL protein preparation, can solve the problems of low yield, high cost, inability to be widely used in vivo experiments or clinical trials, etc., achieve high sensitivity, high specificity, and increase protein yield Effect

Inactive Publication Date: 2011-09-07
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the prior art, the preparation method of 4-1BBL protein is directly extracted from tissue cells, with low yield and high cost
Cannot be widely used in in vivo experiments or clinical

Method used

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  • Preparation method of protein 4-1BBL
  • Preparation method of protein 4-1BBL
  • Preparation method of protein 4-1BBL

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: Preparation of 4-1BBL protein

[0028] (1) Separation of splenocytes from BALB / c mice (the spleens of BALB / c mice were taken aseptically, ground with a glass needle core and passed through a 150-mesh sieve to obtain a single cell suspension), stimulated and cultured in vitro by conA (after washing with RPMI1640, washed with RPMI1640 containing 10% fetal bovine serum (conA5μg / ml) was resuspended and the cell concentration was adjusted to 1×10 6 / ml at 37°C, 5% CO 2 After conditioned culture for 72 h, the cells were collected for extraction of total RNA. ) after trizol lysis (0.1ml cells / 1ml trizol, 42°C for 30-40min), and mRNA was extracted;

[0029] (2) reverse transcription of mRNA (Superscript II reverse transcription kit was purchased from Invitrogen Company) to obtain the cDNA of 4-1BBL extracellular segment protein;

[0030] (3) performing PCR amplification using the cDNA obtained in step (2) as a template to obtain the 4-1BBL protein target gene;

...

Embodiment 2

[0056] Example 2: Immunological activity of 4-1BBL protein

[0057] The 4-1BBL extracellular segment protein obtained by the method of the present invention can effectively enhance the proliferation of T lymphocytes activated by CD3 monoclonal antibody (the relative enhancement rate is 25.6%) and secrete IL-2 after stimulating the mouse T lymphocytes cultured in vitro. (relative enhancement rate is 53.2%), IFN-γ (relative enhancement rate is 65.5%) and other cytokines, showing significant co-stimulatory effect on T cells ( Figure 2 ~ Figure 4 ).

[0058] Method: Mouse splenocytes were filtered through nylon hair column to obtain T lymphocytes, and 10 5 / mL T cells were inoculated in a 96-well plate, and a PBS control group and 3 experimental groups (4-1BBL, anti-CD3+anti-CD28, 4-1BBL+anti-CD3+anti-CD28) were set up, wherein the concentration of 4-1BBL was 1 μg / mL, The concentrations of anti-CD3 and anti-CD28 (purchased from ebioscience) were both 10 μg / mL, and three replica...

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Abstract

The invention provides a preparation method of a protein 4-1BBL. In the preparation method, a recombinant protein 4-1BBL containing 206 amino acid residues (Arg 104-Glu 309) is prepared from an extracellular-segment protein of mouse 4-1BBL (TNFSF9 (Tumor Necrosis Factor Receptor Superfamily 9), NP_033430.1) by using a gene engineering method. By adopting the preparation method provided by the invention, a large quantity of proteins 4-1BBL can be obtained, so that the yield of the protein is greatly improved, and the obtained extracellular-segment protein 4-1BBL can be applied to preparation of kits for the quantitative pre-clinical researches of the protein 4-1BBL or for medicinal purposes and has a wide application prospect.

Description

(1) Technical field [0001] The invention relates to a preparation method of 4-1BBL protein. (2) Background technology [0002] 4-1BB / 4-1BBL is another pair of important co-stimulatory signaling molecules besides CD28 / B7. 4-1BB (CD137) belongs to the tumor necrosis factor receptor (TNFR) superfamily, and is mainly distributed on the surface of activated T cells, NK cells and DC cells, and its ligand 4-1BBL (CD137L) is mainly expressed on professional APC cells ( Such as mononuclear macrophages, DC cells, B cells), activated T cells and some tumor cells. 4-1BBL is a type II transmembrane glycoprotein with a molecular weight of 34KD and 36% homology between humans and mice. [0003] 4-1BB / 4-1BBL signaling plays an important role in enhancing and maintaining immune responses. Compared with the CD28 / B7 signaling pathway, 4-1BB / 4-1BBL is mainly in the late stage of the immune response and is the CD8 + T cell clonal expansion, survival, and memory killing provide signals, and i...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/81C07K14/47
Inventor 黄茵
Owner HANGZHOU NORMAL UNIVERSITY
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