Phage antibody library and application thereof in avian influenza immunodetection

Abstract

The invention relates to an antibody library. An immune globulin light chain variable region VL gene sequence is inserted between Xba and Sac restriction enzyme cutting sites of a Pcomb3 vector. An immune globulin heavy chain variable region VH gene sequence is inserted between Spe and Xho endo restriction enzyme cutting sites. The antibody library is characterized in that two gene fragments comprise the possible combination of antibody variable region light chain and heavy chain, and the antibody library can realize affinity enrichment with an avian influenza virus antigen, and can be applied to avian influenza immunodetection. The antibody library has the advantages that a high affinity antibody can be acquired without animal immunity; a preparation period is short; the antibody library is a phage antibody library which is constructed by taking mice natural immune globulin variable region light chain and heavy chain genes as sources, theoretically contains all possible antibodies in a mice, and is a specificity antibody library which can directly acquire different avian influenza viruses through screening; and the screening has the advantages of high efficiency, high flux, large selection base and high specificity, thus the phage antibody library has a wide application prospect in the aspect of the disease detection of avian influenza and other animals.

Description

Technical field:

[0001] The present invention relates to a phage antibody library and its application, especially an antibody library capable of detecting different subtypes of avian influenza virus and its application in avian influenza immune detection, belonging to an antibody library constructed by phage display technology and It is used in the field of avian influenza immune detection. Background technique:

[0002] Immunological detection technology for bird flu and other animal diseases is a widely used technology to prevent the spread of animal diseases at home and abroad. Together with nucleic acid detection, it constitutes the main method for the prevention and control of animal diseases. Immunoassay has the advantages of simple operation, high sensitivity, strong specificity, and less sample requirement, and has achieved fruitful results in the detection of animal diseases. Among them, the preparation of antibodies is the core content. At present, the preparation...

Examples

Embodiment 1

[0026] Construction of phage antibody library

[0027] Material:

[0028] Five 8-week-old Balb / c mice, weighing 20-25 g, were purchased from Beijing Weitong Lihua Company.

[0029] XL1-Blue competent Escherichia coli cells, antibiotics ampicillin and tetracycline were purchased from Beijing Dingguo Biotechnology Company, Pcomb3 phagemid vector and helper phage VCSM13 were purchased from Pharmacia Company; anti-M13-HRP labeled antibody was purchased from GE-Healthcare Company; Trizol reagent was purchased from Invitrogen; cDNA first-strand synthesis kit, ExTaq DNA polymerase, restriction enzymes (XbaI, SacI, XhoI, SpeI), and T4 DNA ligase were purchased from TaKaRa; DNA gel recovery kit and plasmid extraction kit were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; peptone and yeast extract were purchased from Oxoids.

[0030] Primer design:

[0031] Referring to Parmley et al. (1989), Essono et al. (2003) and database data (Cao Ya, 2003), four sets of pri...

Embodiment 2

[0058] Taking H5N1 influenza virus as an example to screen phage antibodies

[0059] (1) Enrichment and screening of target antibodies

[0060] Using the avian influenza virus as the antigen, the phage antibody library was screened by the direct elution method of the host bacteria, and a total of 4 rounds of affinity elution were carried out.

[0061] (2) Identification of antibodies

[0062] Infect XL1-Blue bacteria with the phage antibody library after 4 rounds of panning, spread 2×YT-AT plate, and then select 20 monoclonal resistance cultures on the plate, after VCSM13 infection, pass through twice PEG (polyethylene glycol) Alcohol) / NaCl precipitation and acetic acid precipitation to purify the recombinant phage, PBS was dissolved and sterilized by filtration through a 0.45 μm filter membrane to prepare the phage recombinant antibody expressed in fusion, and the HRP-labeled anti-M13 monoclonal antibody was used for ELISA identification, and a negative control.

[0063] (...

Technical field:

[0001] The present invention relates to a phage antibody library and its application, especially an antibody library capable of detecting different subtypes of avian influenza virus and its application in avian influenza immune detection, belonging to an antibody library constructed by phage display technology and It is used in the field of avian influenza immune detection. Background technique:

[0002] Immunological detection technology for bird flu and other animal diseases is a widely used technology to prevent the spread of animal diseases at home and abroad. Together with nucleic acid detection, it constitutes the main method for the prevention and control of animal diseases. Immunoassay has the advantages of simple operation, high sensitivity, strong specificity, and less sample requirement, and has achieved fruitful results in the detection of animal diseases. Among them, the preparation of antibodies is the core content. At present, the preparation...

Examples

Embodiment 1

[0026] Construction of phage antibody library

[0027] Material:

[0028] Five 8-week-old Balb / c mice, weighing 20-25 g, were purchased from Beijing Weitong Lihua Company.

[0029] XL1-Blue competent Escherichia coli cells, antibiotics ampicillin and tetracycline were purchased from Beijing Dingguo Biotechnology Company, Pcomb3 phagemid vector and helper phage VCSM13 were purchased from Pharmacia Company; anti-M13-HRP labeled antibody was purchased from GE-Healthcare Company; Trizol reagent was purchased from Invitrogen; cDNA first-strand synthesis kit, ExTaq DNA polymerase, restriction enzymes (XbaI, SacI, XhoI, SpeI), and T4 DNA ligase were purchased from TaKaRa; DNA gel recovery kit and plasmid extraction kit were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; peptone and yeast extract were purchased from Oxoids.

[0030] Primer design:

[0031] Referring to Parmley et al. (1989), Essono et al. (2003) and database data (Cao Ya, 2003), four sets of pri...

Embodiment 2

[0058] Taking H5N1 influenza virus as an example to screen phage antibodies

[0059] (1) Enrichment and screening of target antibodies

[0060] Using the avian influenza virus as the antigen, the phage antibody library was screened by the direct elution method of the host bacteria, and a total of 4 rounds of affinity elution were carried out.

[0061] (2) Identification of antibodies

[0062] Infect XL1-Blue bacteria with the phage antibody library after 4 rounds of panning, spread 2×YT-AT plate, and then select 20 monoclonal resistance cultures on the plate, after VCSM13 infection, pass through twice PEG (polyethylene glycol) Alcohol) / NaCl precipitation and acetic acid precipitation to purify the recombinant phage, PBS was dissolved and sterilized by filtration through a 0.45 μm filter membrane to prepare the phage recombinant antibody expressed in fusion, and the HRP-labeled anti-M13 monoclonal antibody was used for ELISA identification, and a negative control.

[0063] (...

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