Method for rapidly screening SARS-CoV-2 RBD specific fully humanized neutralizing monoclonal antibody

Abstract

The invention belongs to the technical field of monoclone, and particularly discloses a method for rapidly screening SARS-CoV-2 RBD specific fully-humanized neutralizing monoclonal antibody, which comprises the following steps: S1-S3, collecting peripheral blood of SARS-CoV-2 rehabilitation patients, sorting RBD specific memory B cells, obtaining antibody variable region cDNA through RT-PCR amplification; S4, amplifying the antibody variable region cDNA obtained in the steps S1-S3 by adopting nested PCR, and constructing an antibody variable region gene expression box; s5, transferring the antibody variable region gene expression cassette obtained in the step S4 into a cell expression antibody, collecting supernate, and screening an RBD specific monoclonal antibody; and S6, detecting the activity of blocking pseudovirus infected cells by using the RBD specific antibody supernatant obtained in S5, detecting the binding capacity of the supernatant for blocking RBD and ACE2 by using ELISA, detecting the neutralizing capacity of the monoclonal antibody by using a dual method, and screening the RBD specific neutralizing monoclonal antibody. According to the invention, the SARS-CoV-2 RBDspecific fully humanized neutralizing monoclonal antibody can be rapidly and efficiently obtained.

Description

technical field [0001] The invention belongs to the field of monoclonal technology, and in particular relates to a method for rapidly screening a novel coronavirus RBD-specific fully human neutralizing monoclonal antibody. Background technique [0002] Studies have found that the new coronavirus (SARS-CoV-2) has four main structural proteins, namely spike protein (S protein), nucleocapsid protein (N protein), membrane protein (M protein) and envelope protein ( E protein), in which the S protein has two subunits, S1 and S2, and the receptor binding site (RBD) is located on the S1 subunit, its main function is to recognize host cell surface receptors, mediate SARS-CoV-2 and host Fusion of cells. [0003] At present, there is no specific drug targeted treatment for the new pathogen COVID-19, and it will take time to develop a vaccine. The plasma of patients who have been cured and discharged recently contains high concentrations of specific antigen-neutralizing antibodies. Af...

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Problems solved by technology

[0005] The above patent provides a fully human monoclonal antibody whose recognition region is the S1 non-RBD region. However, since the new coronavirus invades host cells through RBD binding to ACE2 of the host cell, the fully human monoclonal antibody obtained in the above patent has no effect on the virus. The blocking effect is limited, and the above-mentioned patent obtains antibody cDNA by labeling plasma cells, and the humoral immune response elicited by plasma cells is limited

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