Expression vector for constructing high-quality bacteriophage antibody library

A carrier and intermediate carrier technology, applied to viruses/bacteriophages, using vectors to introduce foreign genetic material, anti-animal/human immunoglobulins, etc.

Active Publication Date: 2011-06-15
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still many problems to be solved in this technology, such as how to increase the effective library capacity while ensuring the diversity and presentation efficiency of the antibody library, and how to improve the antibody gene connection efficiency when constructing the antibody library. further solution

Method used

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  • Expression vector for constructing high-quality bacteriophage antibody library
  • Expression vector for constructing high-quality bacteriophage antibody library
  • Expression vector for constructing high-quality bacteriophage antibody library

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, the acquisition of carrier PDF-D-SacB

[0037] Escherichia coli trans1-Blue was purchased from Beijing Quanshijin Biological Co., Ltd.; phage VCSM13, BS1365 bacteria, and expression vector pDF were donated by Professor Zhou Lijun of the Naval General Hospital; The plasmid B4-HFLF of human Fab fragment gene of anti-hepatitis B virus surface antigen (HBsAg) is preserved by our laboratory.

[0038] Phusion, BssHII, Xba I, Nco I, Nhe I, BglII, alkaline phosphatase were purchased from New England Biolabs (NEB) company; EcoR I, Dra I, T4 DNA Ligase, T4 Plolynucleotide Kinnase (PNK) were purchased from TaKaRa (Dalian ) company; EasyPure Plasmid MiniPrep Kit and EasyPure Quick Gel Extraction Kit were purchased from Beijing Quanshijin Biological Co., Ltd.; 5000DNA Marker and DL2000DNA Marker were purchased from Beijing Bomaide Company;

[0039] 1. Verify the suicide function of pUC19-SacB

[0040] 1) pUC19-SacB (Wang Yufei, Chen Zeliang, Qiao Feng, Wang Zhoujia, ...

Embodiment 2

[0069] Embodiment 2, the acquisition of phage antibody

[0070] 1. Construction of recombinant plasmids pDF-SacB-HBsL and pDF-SacB-HBsH

[0071] Using plasmid B4-HFLF (Wang Chen, Hou Lihua, Du Guixin, Li Jianming, Chen Puyan, Tong Yigang, construct a preset CDR2 gene phage antibody library to screen anti-human integrin ανβ 3 Humanized Fab of mAb, Journal of Cellular and Molecular Immunology, 2006, 22(1), 64-67; publicly available from Institute of Microbial Epidemiology, Academy of Military Medical Sciences. ) as a template, use two sets of primers in Table 1-2 to amplify the κ chain (primers use VK3-Bgl, IGK-Xba) and Fd segment gene (primers use IGG-Nhe, VH4- r-2Nco1), the reaction conditions are: pre-denaturation at 95°C for 2min; denaturation at 95°C for 20s, annealing at 65°C for 20s, extension at 72°C for 15s, and extension at 72°C for 5min after 35 cycles. The result was: the amplified product of the heavy chain Fd gene was a band of about 680 bp, and the amplified pro...

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Abstract

The invention discloses an expression vector for constructing a high-quality bacteriophage antibody library. The vector disclosed by the invention is prepared by the following steps: 1) inserting coding genes of a light-chain region SacB in multiple cloning sites of pDF to obtain an intermediate vector PSBX; 2) mutating BssHII enzyme cutting sites of the intermediate vector PSBX obtained in the step 1) into BglII enzyme cutting sites to obtain an intermediate vector PSGX; and 3) inserting coding genes of a heavy-chain region SacB in Nhe I and Nco I enzyme cutting sites of the intermediate vector PSGX obtained in the step 2) to obtain a vector pDF-D-SacB. Experiment disclosed by the invention proves that the obtained phagemid vector pDF-D-SacB containing suicide genes is applicable to the construction of a high-capacity bacteriophage antibody library.

Description

technical field [0001] The invention relates to an expression vector constructed and applied to a high-quality phage antibody library. Background technique [0002] The development and application of phage antibody library technology has brought great changes to the field of antibody technology, and has been widely used in various research fields such as biology and medicine. Through the combination of genetic engineering and phage display technology, humanized targeting Antibodies for almost all antigenic epitopes have greatly promoted the development and application of various antibodies with excellent performance and multifunctional antibody fusion proteins. The phage antibody library simulates the natural antibody library, making it possible for people to directly use antigens to screen specific antibodies from the antibody library without going through a complicated immunization process. It not only solves the difficulties in the source of human monoclonal antibody, th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N5/10C12N7/01C07K16/18
Inventor 童贻刚安小平米志强潘博王晓娜张宝中
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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