Biological membrane and preparation method and application thereof

A biofilm and avidin technology, applied in animal cells, vertebrate cells, blood/immune system cells, etc., can solve the problems of lymphocyte cell proliferation multiples and cytotoxic activity, etc., and achieve easy scale production , easy operation, simplified culture procedures and steps

Active Publication Date: 2013-04-03
PERSONGEN ANKE CELLULAR THERAPEUTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is that the proliferation ratio and cytotoxic activity of lymphocyte cells obtained by conventional lymphocyte expansion methods are not ideal, and a method and kit for lymphocyte activation and expansion are provided to solve this problem

Method used

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  • Biological membrane and preparation method and application thereof
  • Biological membrane and preparation method and application thereof
  • Biological membrane and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, the preparation of biofilm

[0038] 1. Construct a fibroblast cell line (Fibroblast-Avidin) stably expressing humanized Avidin (avidin)

[0039] 1) Obtain humanized modified Avidin cDNA (sequence such as GenBank: AJ616762.1) using molecular biology methods, and construct it on the pcDNA3.1 vector, and the recombined vector is named pcDNA-Avidin. The pcDNA3.1 vector was purchased from Invitrogen, the catalog number is V790-20. The specific process is as follows:

[0040] A) entrust Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize the Avidin gene, which has a sticky end cut by Nhe I restriction enzyme at the 5' end and a sticky end cut by Xba I restriction enzyme at the 3' end;

[0041] B) Digest the pcDNA3.1 vector with Nhe I and Xba I (New England Biolabs, Inc, Cat # R0131S and R0145S), recover and purify the digested product (Qiagen, Cat # 28704);

[0042] C) Ligate the synthesized Avidin gene to the digested vector by T4 DNA ligase (Promega, Cat...

Embodiment 2

[0069] Embodiment 2, biomembrane is combined with bioactive molecule

[0070] Biomolecules

[0071] Human Recombinant 4-1BBL, Abnova, Cat# P4065

[0072] human recombinant CD86, Sino Biological Inc. , Cat# 10699-H08H

[0073] Human Anti-CD3 Antibody [OKT3] - Azide free, Abcam , Cat#ab86883

[0074] Reagent

[0075] EZ-Link Sulfo-NHS-Biotin, sigma , Cat# 21217

[0076] operating procedures

[0077] 1) According to the procedure in the reagent manual, label Biotin on human recombinant 4-1BBL, human recombinant CD86, and human anti-CD3 antibody, respectively.

[0078] 2) Wash the labeled biomolecules with PBS.

[0079] 3) Add 10ug of labeled 4-1BBL, CD86, and CD3 antibodies to 2ml of the biofilm prepared above. Incubate at 4oC for 30 minutes.

Embodiment 3

[0080] Embodiment 3, T lymphocyte extraction

[0081] 1) In a 50 ml centrifuge tube, prepare 2×EDTA solution (diluted with DPBS), 25ml per tube.

[0082] 2) Add 25 ml of the obtained blood into the above EDTA solution and mix well.

[0083] 3) Take another 50 ml centrifuge tube and add 25 ml Ficoll solution to each tube.

[0084] 4) Use a pipette to slowly superimpose the diluted blood on the Ficoll solution along the tube wall to keep the interface between the two liquid surfaces clear.

[0085] 5) Tighten the cap of the centrifuge tube and seal it tightly to avoid contamination.

[0086] 6) Centrifugation: The centrifuge model and rotor model are Thermo, IEC CL40R; CAT#11210926, centrifuge at 400g for 30 minutes, speed up to 5 gears, and speed down to 0 gear.

[0087] 7) After centrifugation, the liquid in the tube is divided into three layers, the upper layer is diluted plasma, and the lower layer is mainly red blood cells and granulocytes. The middle layer is Ficoll, a...

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Abstract

The invention provides a separated biological membrane as well as a preparation method and application of the separated biological membrane. The invention further provides a method and an application of the biological membrane for stimulating T lymph cell proliferation; and furthermore, the invention provides a kit containing the biological membrane.

Description

technical field [0001] The invention belongs to the field of biomedicine, especially the field of cellular immunity, and specifically relates to a reagent and a method for activating T lymphocytes and promoting the proliferation of T lymphocytes. The reagent and method can be applied to clinical cell immunotherapy, basic medical research, clinical application research and biotechnology research, especially immune system research and tumor killing effect research. Background technique [0002] T lymphocytes are one of the most important immune cells in the human body. They are differentiated from lymphatic stem cells in the thymus and migrate to the peripheral blood after maturation; they can be divided into three subgroups according to their functions: helper T cells and suppressor T cells and cytotoxic T cells. The normal function of T lymphocytes is extremely important for maintaining the health of the body, and their defects and abnormal functions can lead to various dis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/07C12N5/0783
Inventor 黄纲雄杨林
Owner PERSONGEN ANKE CELLULAR THERAPEUTICS CO LTD
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