Antibody specifically binding to novel coronavirus

A virus and antibody technology, applied in the direction of virus/phage, antibody, virus, etc., can solve the problem of little knowledge about the potential therapeutic application of combined antibodies

Inactive Publication Date: 2021-08-10
THE FIFTH AFFILIATED HOSPITAL SUN YAT SEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, little is known about the potential thera

Method used

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  • Antibody specifically binding to novel coronavirus
  • Antibody specifically binding to novel coronavirus
  • Antibody specifically binding to novel coronavirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Example 1: Expression of SARS-COV-2 viral N protein

[0112] Sequence information (NCBI ID YP_009724397.2) (NCBI ID YP_009724397.2) is obtained from the public data, and the expression of N protein (full length) is synthesized: N-NFL (1-419), N-NTD ( N protein N end, 41-174) and N-CTD (N protein C end, 250-364), for example, figure 1 Indicated. The expression carrier is constructed according to conventional methods to convert suitable host cells to carry out corresponding expression. Briefly, 0.1 mM IPTG was added to the culture medium of the host cell containing the expression vector, and oscillated induced by oscillating culture at 25 ° C, 220 RMP, induced overnight (15h), induced protein expression. The expression of the protein was harvested and the purified protein was isolated by nickel pillars and was subjected to SDS-PAGE electrophoresis. The correct target protein (N protein) was used to screen anti-SARS-COV-2 viral N protein from blood cells from COVID-19 rehabili...

Embodiment 2

[0113] Example 2: Subselect memory B cell

[0114] According to relevant laws and regulations, the 6 volunteers were recruited from the Fifth Affiliated Hospital of Sun Yat-sen University, and the clinical rehabilitation of two throat swab new coronavirus nucleic acid detection is negative, 9-25 days after the onset, collect them Peripheral blood, separated monocytes (PBMC), for example, please refer to the invention patents of the authorization notice number CN107760690B. Serum antibody titer titer detection (ELISA) was performed using purified N protein as an antigen.

[0115] According to the sample antibody titer determined by the ELISA, the highest antibody titer (which is 1: 328050, which is 1: 328050), and a single slurry cell by flow cytometer (BD FACS ARIA III), with CD3 - / CD14- / CD16- / CD235A- / CD19 + / CD20LOW-NEG / CD27HI and CD38HI (BD Biosciences and Invitrogen) set single slurry cells, with CD3- / CD14- / CD16- / CD235A- / CD20- / CD19 + / CD27 + / SARS-COV2 S ...

Embodiment 3

[0116] Example 3: Separation, identification of the variable region gene of the antibody

[0117] Referring to the invention of an invention patent, a suitable primer is designed, and the variable region gene obtained by the screening obtained is amplified, and the corresponding antibody gene sequence is obtained. The specific operation is:

[0118] 1. Anti-transcription synthesis cDNA first chain

[0119] To the 96-well plate containing a single B cell, a constant region primer and a SUPERSCRIPT IV reverse transcriptase (InvitroT IV reverse transcriptase) were added to the light chain, respectively, 37 ° C for 1 hour in 96-well plates containing a single B cell.

[0120] 2. Separate antibody gene by two-wheel PCR program

[0121] The first round of PCR: 50 ul system contains 5 ul of reverse transcription reaction products, 25 μl of TAQ MIX (Invitrogen, Carlsbad, Ca), and 0.5 um of each sub-heavy chain and light chain antibody constant region primers, PCR reaction conditions: The ...

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Abstract

The complement activation is a risk factor for the morbidity and mortality of novel coronavirus-19 (COVID-19) patients, and is mediated by a highly immunopathogenic nucleocapsid protein (N) in which severe acute respiratory distress syndrome-related coronavirus 2 (SARS-CoV-2) binds to serine protease MASP-2 in the agglutinin pathway of complement activation. According to the invention, a dominant antibody with anti-SARS-CoV-2 virus N protein is separated and identified from a rapidly recovered COVID-19 convalescent person, and the antibody has high binding affinity. In addition, based on the cleavage activity of the complement protease MASP-2 on a specific fluorescence quenching peptide substrate, the invention further develops a virus-free complement hyper-activation analysis method.

Description

Background technique [0001] RNA viruses such as SARS-COV-2, SARS-COV, ZIKA, MERS-COV, EBOLA, H7N9 are popular in local area / world, and bring serious hazards to people's life and health. And the economic development of the world brings huge impact. [0002] The coronavirus (SARS-COV) is a film-enveloped virus (the lipid double layer derived from the hosted cell membrane), has a mainly viral protein (such as a spicy protein (SPIKE, S), membrane protein (MEMBRANE) , M), envelope protein (E), Nucleocapsid, N)) formed viral structure, wherein S protein, M protein and E protein are embedded in a viral envelope, N protein and virus RNA Combined with the genome package in riboconuclein particles. Different from probular proteins (s) having a certain mutation frequency, the sequence of nuclear casing proteins is more stable, which means it is an ideal target for diagnostic tools and antiviral therapy. [0003] The complement is one of the first defense lines of congenital immunization, a...

Claims

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Application Information

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IPC IPC(8): C07K16/10A61K39/42A61K47/68A61P31/14G01N33/68G01N33/577G01N33/569C07K14/165
CPCC07K16/10A61K47/6817A61K47/6841A61P31/14G01N33/6854G01N33/577G01N33/56983C07K14/005C07K2317/565C07K2317/56C07K2317/34C07K2317/92C07K2317/76A61K2039/505G01N2333/165G01N2469/20C12N2770/20022
Inventor 单鸿陈守登廖化新郑伟宏肖非康斯斯
Owner THE FIFTH AFFILIATED HOSPITAL SUN YAT SEN UNIV
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