All-human-sourced novel coronavirus IgG2 single-chain antibody and application thereof
A single-chain antibody, fully human technology, applied in the field of biomedicine
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Embodiment 1
[0045] Example 1: Full-length amplification and sequencing of BCR in recovered patients and normal population
[0046] The full-length amplification and sequencing of BCR of the recovered COVID-19 patients and the normal population was carried out according to the patented method (publication number CN111662970A) previously applied for. The main process is as follows:
[0047] 1. Extraction of Total RNA from whole blood samples
[0048] Take 1 mL of fresh whole blood sample, use TriZol LS to extract the total RNA of the whole blood sample, use Nanidrop 2000C to measure the concentration and purity of RNA sample after extraction, use Agilent 2100 to measure the integrity of the sample, and the samples that meet the qualified standard (total amount > 1 μg , Integrity RIN value > 7) for subsequent experiments.
[0049] 2. Synthesis of first-strand cDNA from total RNA
[0050] The experimental operation process is as follows:
[0051] 1) The Oligo dT reverse transcription prime...
Embodiment 2
[0095] Example 2: Analysis of the BCR immune group library of recovered COVID-19 patients and normal population to obtain COVID-19-specific IgG2 single-chain antibody
[0096] After performing the HiFi consistency correction on the raw sequencing data, a BCR full-length consensus sequence with a quality value above Q20 was obtained, and after comparison with the antibody constant region sequence in the BCR database, the sequencing data of each sample was obtained Different classes of BCR antibody sequences in the DNA sequence are translated into protein polypeptide sequences based on the stop codon position of the constant region. By comparing the BCR polypeptide sequences shared by the recovered patients and not in the normal population, the full length of the new crown-specific antibodies was obtained. The 12 new crown-specific IgG2 sequences obtained this time, all of which include the VDJ region, include the start codon ATG at the 5' end, and include the stop codon TGA at ...
Embodiment 3
[0097] Example 3: Construction of a monoclonal BCR antibody library based on the design of specific primers based on samples from recovered patients
[0098] Based on the IgG2 antibody sequence screened by the antibody library, specific primers were designed outside the start codon and stop codon, and the target antibody sequence was specifically enriched by PCR amplification. Due to the multiple recombination characteristics of antibodies, the obtained amplified products are a set of antibody sequences matched with primers, which cannot be directly used for first-generation sequencing and subsequent applications. It is necessary to perform cloning experiments on antibody sequences to obtain monoclones of individual antibody sequences, and Select a single clone for sequencing, and verify that the obtained monoclonal sequence is the target antibody. The specific process is as follows:
[0099] 1. Target antibody IgG2 antibody sequence primer design
[0100] Design primers bas...
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