Phage antibody library and application thereof in avian influenza immunodetection
A phage antibody library, avian influenza technology, applied in the antibody library constructed by phage display technology, avian influenza immune detection field, to achieve the effect of strong specificity, high throughput and high efficiency
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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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