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Cloning for sifting tumor hecrosis target antibody, preparing method and use thereof

A technology targeting antibodies and tumor necrosis, applied in the field of biomedicine, can solve the problems of screening TNT antibody clones and preparation methods that have not yet been seen, and achieve the effects of easy development, simple process, and high probability

Inactive Publication Date: 2005-02-23
上海晨健抗体组药物有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, so far, there have been no new reports on cloning and preparation methods for screening TNT antibodies

Method used

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  • Cloning for sifting tumor hecrosis target antibody, preparing method and use thereof
  • Cloning for sifting tumor hecrosis target antibody, preparing method and use thereof
  • Cloning for sifting tumor hecrosis target antibody, preparing method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1. A selection of donors and RNA extraction

[0094] 1. Screen patients

[0095] ① Among hospitalized patients with autoimmune diseases, select 6 patients with strong autoantibody positive, and the standard is that the autoantibody titer is greater than 1:1,000;

[0096] ②Perform bone puncture, draw 3ml of bone marrow fluid separately, and dilute by phosphate buffered saline (PBS for short);

[0097] 2. Lymphocyte separation liquid separation method routinely separates mononuclear cells

[0098] ①Take 5ml of lymphocyte separation solution (Shanghai Chemical Reagent Factory, analytical grade) and place it in a sterile centrifuge tube;

[0099] ② Spread the diluted bone marrow fluid lightly on the surface of the separation liquid with a straw;

[0100] ③ Centrifuge in a horizontal centrifuge, 2,000 rpm (revolutions per minute), 20 minutes;

[0101] ④Aspirate the cells in the lower cell layer of serum and wash 3 times with PBS;

[0102] ⑤Adjust the cell concentration f...

Embodiment 2

[0108] Example 2. Alternative donor selection and RNA extraction

[0109] 1. Screen patients

[0110] ① Among hospitalized patients with autoimmune diseases, select 3 patients with multiple autoimmune diseases and strong autoantibodies. The standard is that the autoantibody titer is greater than 1:10,000;

[0111] ②Perform bone puncture, draw 3ml of bone marrow fluid separately, and dilute by phosphate buffered saline (PBS for short);

[0112] 2. Isolation of mononuclear cells by immunomagnetic bead separation cell method

[0113] ① Suspend the immunomagnetic bead solution sufficiently (without magnetic field);

[0114] ②Take Tosyl, 0.1ml of activated magnetic beads (M-450, Dynal);

[0115] ③Place it under the magnetic field for 4 minutes;

[0116] ④B takes out the test tube, removes the supernatant, and washes with B buffer solution twice;

[0117] ⑤Dilute the antigen (nucleoprotein component) with B buffer to 0.1ml (about 30 micrograms / ml);

[0118] ⑥ Suspend the magnetic beads ...

Embodiment 3

[0131] Example 3. Amplification and cloning of TNT antibody gene

[0132] 1. Design and synthesis of primers for antibody gene amplification

[0133] According to relevant literature reports in the past, the ratio of IgG1 to IgG3 in autoantibodies is the highest, so you can refer to conventional laboratory manuals and other literature to design primers as follows:

[0134] The specific primer sequence of VH+CH1(IgG1) is:

[0135] 5’ forward primer: 5’-CAG GTG CAG CTG CTC GAG TCG GG

[0136] 3’ end reverse primer: 5’-GCA TGT ACT AGT TTT GTC ACA AGA TTT GGG

[0137] The specific primer sequence of VH+CH1(IgG3) is:

[0138] 5’ forward primer: 5’, CAG GTG CAG CTG CTC GAG TCG GG

[0139] 3’ end reverse primer: 5’-TGT GTG ACT AGT GTC ACC AAG TGG GGT TTT

[0140] The specific primer sequence of VL+CL(κ) is:

[0141] 5’ end forward primer: 5’-GAA ATT GAG CTC ACG CAG TCT CCA

[0142] 3’ end reverse primer: 5’-GCG CCG TCT AGA ATT AAC ACT CTC CCC TGT TGA

[0143] The specific primer seq...

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Abstract

A clone for screening tumor necrosis target antibody, its preparing method and use are disclosed. It includes: selecting autoimmune disease patient, extracting B lymph cell and phlogocyte with high-expressing auto-antibody, extracting RNA, amplifying and cloning antibody gene, constructing superficial displaying library by Helper protobiology, multiple screening this library, removing gpIII gene fragment, converting competent bacteria dissolving expressing after re-connecting, selecting single-cloning antibody and enriching expressing, selecting positive clone for screening tumor necrosis target antibody. It achieves low cost, simple operation and rapid industrial method.

Description

Technical field [0001] The present invention relates to the field of biomedicine technology, in particular to clones used for screening monoclonal antibodies and preparation methods and uses thereof, and more specifically to clones used for screening tumor necrosis targeting antibodies and preparation methods and uses thereof . Background technique [0002] Molecular targeted therapy of tumors is the most dynamic and concerned field in recent years. It is a tumor-specific treatment method in a fundamental sense. The core of its technology is targeted molecules. People can choose any tumor-killing substance and target. Molecular binding. An ideal targeting molecule should have the following characteristics: ①High specific binding to the target antigen; ②In the appropriate molecular weight range, so that the targeting molecule can penetrate the tumor tissue; ③When it binds to the target molecule, it exhibits high affinity; ④The chemical structure of the molecule is useful for stabi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C07K16/30C12N7/01C12N15/13G01N33/53
Inventor 韩焕兴倪健杨子义何玮
Owner 上海晨健抗体组药物有限公司
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