Bovine-derived single-chain antibody for resisting staphylococcus aureus LukD virulence factor and preparation and application thereof
A technology of single-chain antibody and staphylococcus, applied in application, antibody, anti-bacterial immunoglobulin, etc., can solve the problem of no anti-staphylococcus aureus and achieve the effect of inhibiting the pathogenic activity of staphylococcus aureus
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Embodiment 1
[0054] Example 1 Construction of bovine phage single-chain antibody library
[0055] 1. Collect the blood of dairy cows suffering from mastitis. When the ELISA method detects that the serum antibody titer is greater than 1:20000, continue the follow-up experiment. Bovine peripheral blood leukocytes were extracted with anticoagulated blood, and total RNA was extracted by Trizol method (TRIZOL Reagent was purchased from TaKaRa Company). Using the extracted total RNA as a template, Oligo primer was used to synthesize the first-strand cDNA according to the product instructions of the reverse transcription kit (cDNA first-strand synthesis kit was purchased from TaKaRa Company).
[0056] 2 Analyze the variable region sequence of the bovine antibody coding gene in the published literature, and design primers for amplifying the light and heavy chains of the antibody (Table 1) according to the FR region, wherein VH F and VH R are used to amplify the VH region; VL F and VL R are used t...
Embodiment 2
[0063] Example 2 Screening of bovine-derived anti-Staphylococcus aureus virulence factor LukD single-chain antibody
[0064] 1 Enrichment and panning to prepare the F component (LukE) of the two-component leukocidin LukED of Staphylococcus aureus (ATCC25923), use it as an antigen, and coat overnight at 4°C; block 96 wells with PBST containing 4% skimmed milk powder Plate, incubate at 37°C for 2h; add the single-chain antibody phage antibody library prepared in the above steps to the 96-well plate, incubate at 37°C for 2h, wash with PBST and PBS 10 times each, and wash off unbound free phage; each well Add 100ul 0.2mol / L Gly-Hcl buffer (PH=2.2) to elute the specifically bound phage, add 50ul 1mol / L Tris-Hcl (PH=9.1) to neutralize the eluate; infect the remaining part of the eluate After E. coli TG1, repeat the above steps. Repeat this for 3-5 rounds. After the first round, the stringency of washing should be increased: wash with PBS 20 times after elution with PBST for 20 time...
Embodiment 3
[0066] Example 3 Prokaryotic expression and purification of single chain antibody pGEX-4T-1-scFv-22
[0067] 1 Construction of the recombinant plasmid pGEX-4T-1-scFv Using the No. 22 positive clone strain as a template, use specific primers (as shown in Table 2) to amplify the scFv-22 target gene, and select restriction enzymes EcoR I and Xho I double-enzyme-digest the target gene and the prokaryotic expression vector pGEX-4T-1, connect to obtain the recombinant plasmid after digestion, transform it into DH5α competent, colony PCR and double-enzyme digestion of the plasmid verify that the correct clone is sent to Shanghai Boshang Biotechnology Limited Sequencing;
[0068] Table 2 Primers for amplifying single-chain antibodies and the size of their amplified fragments
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[0071]The clones with correct sequencing were extracted from the plasmids, and then the recombinant plasmids were transformed into BL21 competent cells, and single clones were picked, co...
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