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Antitumor human single-chain antibody

A technology with anti-tumor and anti-tumor effects, which is applied in the direction of anti-tumor drugs, antibodies, anti-enzyme immunoglobulins, etc., can solve the problems of lack of specificity, toxic and side effects, unstable RNA interference technology, narrow range of action, etc., and achieve immunogenic Low stability, small molecular weight, stable combination effect

Active Publication Date: 2011-08-03
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In view of the lack of specificity of the above-mentioned small molecule inhibitors that inhibit CDK4 activity, the toxicity and side effects caused by tumor cell targeting, and the relative instability and narrow range of action of antisense technology and RNA interference technology, the purpose of the present invention It is to provide a low-toxic, efficient, specific and stable active molecule that inhibits CDK4, that is, anti-CDK4 human single-chain antibody, and realize its phenotype function of effectively knocking out CDK4 in cells, so as to inhibit the proliferation of tumor cells Purpose

Method used

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  • Antitumor human single-chain antibody

Examples

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Effect test

Embodiment 1

[0033] Example 1 Preparation of specific anti-human CDK4 human single-chain antibody, for ease of reading, the patent "specific anti-human CDK4 human single-chain antibody" hereinafter referred to as "ANK4"

[0034] 1. Experimental materials

[0035] 1. Plasmids and strains

[0036] Escherichia coli (Escherichia coli) DH5α, HB2151 and XL1-Blue were purchased from Beijing Dingguo Biotechnology Co., Ltd. The pUC119 plasmid was purchased from Dalian Bao Biological Engineering Co., Ltd. Helper phage VCSM13 was purchased from Stratagene.

[0037] 2. Main related reagents for molecular cloning

[0038] Tryptone and yeast extract for bacterial culture were purchased from Oxid Company. The recombinant human CDK4 protein expressed in prokaryotic and purified by Ni-NTA Agarose was prepared according to the method of Cao Yuhua et al. in Jilin University Journal (Natural Science Edition), 2008, 46(5): 992-996 in 2008; HRP / Anti-M13 monoclonal antibody Purchased from Pharmacia Company,...

Embodiment 2

[0057] Example 2 Characterization of the activity of the anti-CDK4 human single-chain antibody ANK4

[0058] 1. Experimental materials

[0059] The pET28a-CDK4 / BL21(DE3) strain was prepared according to the method of Cao Yuhua et al. in Jilin University Journal (Natural Science Edition), 2008, 46(5): 992-996 in 2008. The human breast cancer MCF-7 cell line was provided by the Department of Immunology, Jilin University. DMEM for cell culture and calf serum were purchased from Gibco. DTT, Trypsin, Protein G on 4B fast flow was purchased from Sigma, anti-β-actin mouse monoclonal antibody was purchased from Sigma, rabbit anti-CDK4 polyclonal antibody was purchased from Santa Cruz Biotechnology, and HRP-labeled goat anti-rabbit IgG was purchased from Beijing Dingguo Biotechnology Co., Ltd. company. Cell culture plates, culture flasks, and cell scrapers were purchased from Costar. Refer to Example 1 for the rest of the reagents and materials.

[0060] 2. Experimental method ...

Embodiment 3

[0086] Example 3 Cloning of anti-CDK4 human intracellular single-chain antibody gene and construction of recombinant eukaryotic expression vector. For ease of reading, the patent "anti-CDK4 human intracellular single-chain antibody gene" is hereinafter referred to as "NANK4"

[0087] 1. Experimental materials

[0088] 1. Strains and Plasmids

[0089] pcDNA3.1 (+) plasmid was purchased from Invitrogen; Escherichia coli E.coli JM109 was purchased from Beijing Dingguo Biotechnology Co., Ltd.

[0090] 2. Main related reagents for molecular cloning

[0091] Restriction enzymes Hind III, EcoR I, T4 DNA ligase and its corresponding buffer, Taq DNA polymerase and its corresponding buffer, and dNTP were purchased from Dalian Bao Biological Engineering Co., Ltd. Plasmid extraction and purification kits were purchased from Promega. DNA recovery kit, nucleic acid molecular weight standard (1kb Ladder and DL-2000DNAMarker) were purchased from Beijing Dingguo Biotechnology Co., Ltd. Ag...

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Abstract

The invention relates to an antitumor human single-chain antibody, which belongs to a human antibody, in particular to a specific anti-human D1-cyclin-dependent kinase 4 (CDK4) human single-chain antibody which has a nucleotide sequence represented by a Sequence No.1. The antitumor human single-chain antibody also comprises genetic coding sequence of nuclear localization signal (NLS) peptide and an E-tag peptide, and the nucleotide sequence of the genetic coding sequence is represented by a Sequence No.2. The invention also relates to the use of the antitumor human single-chain antibody in the preparation of antitumor medicines, wherein the tumors treated are human breast cancer and human cervical carcinoma. The invention antitumor human single-chain antibody is a low-toxicity, high-efficiency and specific molecule for inhibiting the activity of CDK4 and has the advantages of small molecular weight, low immunogenicity and the like. A tumor specific eukaryotic expression vector is usedto perform the tumor targeted treatment of the antibody and realize the controllability of the therapeutic gene, and the tumor specific eukaryotic expression vector has tumor specificity and therefore ensures treatment safety and effectiveness.

Description

technical field [0001] The present invention relates to a human antibody. Specifically, it is the gene acquisition and activity characterization of a human single-chain antibody that specifically recognizes CDK4 protein with anti-tumor activity. Background technique [0002] Tumor is the main killer that threatens human life and health, and currently there is no effective treatment. [0003] One of the main reasons for the malignant proliferation of tumor cells is the uncontrolled cell cycle. Evan GI et al. proposed in Nature, 2001, 411: 342-348 in 2001 that the core of the cell cycle regulation mechanism is the phasic activation of cyclin-dependent protein kinases (cyclin-dependent kinases, CDKs). CDKs are a class of Ser / Thr protein kinases whose activity depends on the combination with Cyclin. Abnormal activation of CDKs will lead to uncontrolled cell cycle, thereby inducing tumor formation. Malumbers M et al. summarized in Trends in Biochemical Sciences, 2005, 30(11):...

Claims

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Application Information

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IPC IPC(8): C07K16/40A61K39/395A61P35/00
Inventor 李桂英王磊许晶晶冯晶
Owner JILIN UNIV
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