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Flavivirus antibodies and uses thereof
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A flavivirus and antibody technology, applied in the field of biomedicine, can solve the problems of complicated instruments, time-consuming and high cost, and achieve the effect of high affinity, high application value and accurate detection
Active Publication Date: 2021-11-23
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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At present, the most reliable diagnostic method is to extract viral RNA and perform qRT-PCR. This method is time-consuming, expensive, and needs to be operated by professionals in a specific laboratory. The instruments used are also complicated and cannot be widely and conveniently applied.
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[0038] 1. Expression and purification of yellow fever virus nonstructural protein 1 (YFV-NS1)
[0039] The extracellular region gene fragment of YFV-NS1 was cloned into the pFastBac I vector through Nde I and Xho I, and transformed into Escherichia coli DH10 to obtain the recombinant plasmid Bacmid-NS1. Then, sf9 cells were transfected to obtain baculovirus containing NS1 gene. After baculovirus infects Hi5 cells, it can secrete and express NS1 protein into the culture supernatant. The protein was purified by affinity chromatography and molecular sievechromatography, and the protein purity was identified by SDS-PAGE. The results show that the NS1 protein exists in the form of monomers and dimers, and the size of the monomer is 43kDa (as shown in SEQ ID NO: 23). The results are as follows figure 1 . Concentrate YFV-NS1 to 1 mg / mL and store it frozen at -80°C for future use.
[0040] Other flaviviruses WNV-NS1, ZIKV-NS1, DEN...
[0051] 1. ELISA detection of antibody binding activity
[0052] 1) Dilute antigen 2μg / mL (YFV-NS1, YFV-NS1 C terminal, WNV-NS1, ZIKV-NS1, DENV1-NS1, DENV2-NS1, DENV3-NS1, DENV4-NS1) with coating solution, add to enzyme In the target plate, 100 μL / well, overnight at 4°C.
[0053] 2) The next day, add 200 μL / well of PBST, wash the plate 5 times with a plate washer (BIO-TEK, 405LS), add 5% skimmed milkpowder blocking solution, 100 μL / well, and incubate at 37° C. for 1 hour.
[0054] 3) Rinse 5 times with PBST, add antibody YB31, YB44, YD4 or YD33, 400ng / well, incubate at 37°C for 1 hour.
[0055] 4) Rinse 5 times with PBST, HPR-labeled goat anti-human IgG, 100 μL / well, and incubate at 37° C. for 45 minutes.
[0056] 5) Rinse with PBST for 5 times, add TMB chromogenic solution, 100 μL / well, keep at room temperature in the dark, react for 2 minutes, then add 2M H2SO 4 Stop the reaction, 100 μL / well.
[0057] 6) Detect...
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Abstract
The invention relates to flavivirus antibodies and uses thereof. The flavivirusantibody comprises a heavy chain variable region and a light chain variable region, and amino acid sequences of the heavy chain variable region and the light chain variable region are shown in a sequence table. The invention also discloses nucleotide acid sequences for coding the heavy chain variable region and the light chain variable region, application of the antibody to preparation of drugs or reagents for detecting, treating and / or preventing flavivirus, and a pharmaceutical composition or reagent containing the antibody. The antibody has high affinity to flavivirus.
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