Neutralizing human monoclonal antibody against novel coronavirus and its application
A coronavirus and antibody cloning technology, applied in the direction of antibodies, applications, antiviral agents, etc., can solve the problems of no specific antiviral drugs, no effective preventive vaccines, etc., to achieve good neutralization activity, prevention and treatment of diseases Effect
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Embodiment 1
[0042] Example 1: Expression and purification of novel coronavirus receptor binding domain (RBD) protein
[0043]The amino acid sequence (319-541) of the extracellular domain of the spike glycoprotein (S protein) gene sequence of the new coronavirus SARS-CoV-2 (GenBank: QHR63250.2) and the N-terminal signal peptide derived from albumin, the C-terminal thrombin site The Fc fragment tag of the dot and human IgG1 was constructed into the pSectag2A vector. The specific process was to convert the receptor binding domain (RBD, amino acid from 319 to 319) in the envelope S protein of the novel coronavirus (GenBank: QHR63250.2). After the gene fusion of 541) (WH-RBD), thrombin site (T) and human IgG1 Fc fragment (Fc), the enzyme was constructed into the pSecTag2A vector (ThermoFisher Scientific, Cat. No. V90020) by homologous recombination. At the cut site SfiI, the vector pSectag2A-WH-RBD-TFc was successfully obtained. It was expressed using mammalian cell 293F expression system. O...
Embodiment 2
[0046] Example 2: Screening using a phage Fab display library
[0047] Using the antibody Fab phage display library, the receptor binding domain of the novel coronavirus spike glycoprotein (S) expressed in the mammalian cell 293F expression system was used as the antigen, and the phage Fab library was panned by immunomagnetic bead method. Antigen capture, washed with PBS+0.05% Tween-20, after 4 rounds of screening, and single clone identification and sequencing, a clone with neutralizing activity on a live virus in vitro was obtained, named Fab-F1-A3.
[0048] Sequence listing of Fab-F1-A3
[0049]
Embodiment 3
[0050] Example 3: Expression and purification of Fab-F1-A3
[0051] Fab-F1-A3 was expressed and purified according to the existing literature (Zhu Z, Dimitrov DS. Methods Mol Biol. 2009; 525: 129-4). The Fab-F1-A3 prokaryotic expression vector was constructed and transformed into E.coli HB2151. Then inoculate the strains in SB medium containing 100 μg / ml ampicillin (1L medium contains 30 g tryptone, 20 g yeast extract and 10 g MOPS, and the pH value is adjusted to 7.0 with NaOH), and add when the OD600 reaches 0.7~1.0. The final concentration of IPTG was 200 μg / ml, and expression was induced under the conditions of 37°C and 220rpm for 14-16h. The cells were collected by centrifugation at 4°C, 6000 rpm, 15 min, the medium was discarded, the pellet was resuspended in 1×PBS, and then treated with polymyxin B (polymyxin B) for 45 min, and the supernatant was collected by centrifugation. It was purified with Ni-NTA filler and its purity was verified by SDS-PAGE. The buffer was t...
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