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Leukemia single-chain antibody library, as well as construction method and application thereof

A single-chain antibody, leukemia technology, applied in the field of genetic engineering, can solve the problems of immunogenicity, problems, limitations, etc., and achieve the effect of large library capacity, good diversity, and strong operability

Active Publication Date: 2013-03-20
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The first generation of mouse monoclonal antibody used in the treatment of human diseases is mainly mouse-derived monoclonal antibody, but the main disadvantage of mouse monoclonal antibody is that it causes the human immune response, and the human anti-mouse antibody produced by the human body makes it clear quickly and cannot play its biological role well Therefore, the clinical application is limited; the second-generation antibody is based on the mouse monoclonal antibody and forms a chimeric antibody and a humanized antibody through gene recombination, but there are still immunogenicity problems when the two are applied to the human body; At present, people are working on the research of the third-generation antibody, that is, fully human monoclonal antibody, mainly using phage display technology (phage display) combined with genetic engineering technology, and the resulting multiple strains of human monoclonal antibody have been successfully produced Applied to the treatment of clinical diseases

Method used

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  • Leukemia single-chain antibody library, as well as construction method and application thereof
  • Leukemia single-chain antibody library, as well as construction method and application thereof
  • Leukemia single-chain antibody library, as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Extraction of Total RNA and Synthesis of cDNA in Lymphocytes of Leukemia Patients

[0032] experimental method:

[0033] (1) Separation of blood lymphocytes from leukemia patients: Take the blood of leukemia patient volunteers, use TAKARA company's lymphocyte separation medium, and separate peripheral blood lymphocytes according to the following methods for each:

[0034] ①Take 1-2ml of peripheral blood from leukemia patients and add an equal volume of PBS to dilute;

[0035] ②Take a sterile 15m1 centrifuge tube, add lymphocyte separation solution, make the separation solution: PBS: peripheral blood = 1:1:1, carefully add the diluted blood to the separation solution, try to get close to the liquid surface at the beginning, slowly slow drop;

[0036] ③ Place in a low-speed horizontal centrifuge, 2500rpm, room temperature, and centrifuge for 20 minutes. During the centrifugation process, the speed up and down should be slow to avoid mixed layers;

[0037] ④T...

Embodiment 2

[0062] Example 2: Amplification of Antibody Heavy Chain and Light Chain Variable Region Genes by PCR

[0063] experimental method:

[0064] (1) Primer design:

[0065] According to the human antibody gene sequence in GenBank, the primers required for PCR amplification of antibody heavy chain and light chain variable regions were designed respectively (see Table 1), a total of 32 (sequences are shown in SEQ ID NO.1-32), entrusted Synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0066] Table 1. Primers for PCR amplification of human immunoglobulin variable region genes

[0067]

[0068]

[0069] Note: B=C,G;K=G,T;M=A,C;R=A,G;S=G,C;W=A,T;Y=C,T

[0070] (2) amplify heavy chain variable region (VH) and light chain variable region (VL) with PCR method:

[0071] ① Add the following reagents in turn to the PCR tube:

[0072]

[0073] ②Take another PCR tube, replace the cDNA template with sterile double-distilled water, and use the same reagents as the blank cont...

Embodiment 3

[0079] Example 3: Assembly and full-length amplification of single-chain antibody (scFv) gene fragments

[0080] experimental method:

[0081] 1) Design of linker and primers with restriction enzyme sites

[0082] The separately amplified VH gene fragment and VL gene fragment are connected through a linker sequence to form a scFv gene. In this experiment, the linker designed VH and VL variable region primers according to the scheme of (GGGGS) 3, respectively added SfiI and NotI restriction sites and several protective bases at the 5' end, and added the linker sequence at the 3' end (see Table 2, the sequence is shown in SEQ ID NO.33-64).

[0083] Table 2. List of primers with junction regions and restriction enzyme sites

[0084]

[0085]

[0086]

[0087] Note: ①The linker sequence and restriction site are underlined in italics; ②In the table: B=C,G;K=G,T;M=A,C;R=A,G;S=G,C;W =A,T;Y=C,T

[0088] 2) PCR amplification of the VH and VL fragments that introduce part ...

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Abstract

The invention provides a leukemia bacteriophage single-chain antibody library, which uses phasmid pCANTA-5E as a carrier, has storage capacity of 1.5*10<9>, and has excellent diversity in proving of DNA (deoxyribonucleic acid) sequencing. A leukemia cell specific completely humanized single-chain antibody can be obtained conveniently in multiple screenings by using a specific leukemia related antigen as a target through a bacteriophage representation technology from the completely humanized single-chain antibody library. The antibody library provided by the invention has high storage capacity and excellent diversity, and comprises a combination of complete humanized single-chain antibodies; and a single-chain antibody is formed by connection of a heavy chain variable region VH and a light chain variable region VL through a connecting peptide (GGGGS)3, and contains a complete antigen-binding site. The method has the advantages of strong operability and high storage capacity, and can be used for conveniently screening the leukemia cell specific completely humanized single-chain antibody; and the completely humanized single-chain antibody can be used for preparing a target medicament, which is used for treating leukemia.

Description

technical field [0001] The invention belongs to genetic engineering, and relates to a fully human leukemia single-chain antibody library and its construction method, and its application in the preparation of leukemia antibody medicine. Background technique [0002] since Since he and Milstein founded lymphocyte hybridoma technology, monoclonal antibodies have been widely used in the field of tumor diagnosis and treatment. The first generation of mouse monoclonal antibody used in the treatment of human diseases is mainly mouse-derived monoclonal antibody, but the main disadvantage of mouse monoclonal antibody is that it causes the human immune response, and the human anti-mouse antibody produced by the human body makes it clear quickly and cannot play its biological role well Therefore, the clinical application is limited; the second-generation antibody is based on the mouse monoclonal antibody and forms a chimeric antibody and a humanized antibody through gene recombinatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B40/10C12N15/13C12N15/63A61K39/395A61P35/02
Inventor 詹金彪范娜娜张珍珍林莉汤沁代争
Owner ZHEJIANG UNIV
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