Camel-source high-affinity nano antibody for SARS-CoV-2alpha mutant strain and SARS-CoV-2beta mutant strain

A sars-cov-2, antibody technology, applied in the direction of antibodies, antiviral agents, chemical instruments and methods, etc., can solve the problems of difficult to obtain plasma of patients who have recovered from serum therapy, and the amount is small, and achieves simple pretreatment process, high neutralization Ability, high sensitivity effect

Pending Publication Date: 2022-05-27
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
0 Cites 0 Cited by

AI-Extracted Technical Summary

Problems solved by technology

However, the limitation of patient serum therapy is that it is difficult to obtain the plasma of recovered patients, and the quantity is...
View more

Abstract

The invention relates to a camel source high-affinity nano antibody of SARS-CoV-2 alpha mutant strain and beta mutant strain, in particular to an antibody specifically bound with S protein of coronavirus (such as SARS-CoV-2) and an antigen binding fragment of the antibody. More specifically, the present invention relates to camel-derived nanoantibodies or antigen-binding fragments thereof capable of binding with high affinity to surface S proteins of coronaviruses such as SARS-CoV-2 wild-type original strains and mutants thereof (especially B.1. 1.7 mutant strains and B.1.351 mutant strains), which can be used for the prevention, detection, diagnosis or treatment of infections caused by coronaviruses, especially SARS-CoV-2 viruses.

Application Domain

Immunoglobulins against virusesAntivirals +2

Technology Topic

AntigenMutant strain +8

Image

  • Camel-source high-affinity nano antibody for SARS-CoV-2alpha mutant strain and SARS-CoV-2beta mutant strain
  • Camel-source high-affinity nano antibody for SARS-CoV-2alpha mutant strain and SARS-CoV-2beta mutant strain
  • Camel-source high-affinity nano antibody for SARS-CoV-2alpha mutant strain and SARS-CoV-2beta mutant strain

Examples

  • Experimental program(7)

Example Embodiment

[0075] Example 1. Construction of SARS-CoV-2 Nanobody Library
[0076] Take 200ug of SARS-CoV-2 virus original strain S protein and RBD protein (Beijing Yiqiao Shenzhou Biological Co., Ltd.) and mix it with an equal volume of complete Freund's adjuvant, fully emulsify and inject it into camels, and then boost the immunization every two weeks. Among them, the mixture of incomplete Freund's adjuvant and immunogen was used in booster immunization, and subcutaneous immunization on the back of the neck was performed at multiple points for a total of 5 times. From the third immunization, blood was collected from the jugular-clavicular vein one week after each immunization and serum titers were detected.
[0077] Leukocytes were isolated from the peripheral blood after the fifth immunization, total RNA was extracted, and the VHH gene was cloned by reverse transcription PCR and nested PCR (wherein, the systems and parameters of reverse transcription PCR and nested PCR are described below) Fragment, the sticky ends were modified with the restriction endonuclease SfiI, the VHH gene fragment was ligated to the phagemid pComb3Xss by T4 ligase (a gift from the laboratory of Prof. Bruce D Hammock, UC Davis), and the high-efficiency electroporation was transformed into E. coli ER2738 (laboratory) Preservation, or commercially available, for example, from NEB, UK), to construct a phage nanobody library of SARS-CoV-2. It has been determined that the primary library has a capacity of 10 9cfu, add helper phage (multiplicity of infection: 20:1) M13KO7 (purchased from NEB Company, product number: N0315S) for rescue, and obtain a phage nanobody library with a library capacity of 10 12 pfu/mL, the diversity of the library is better.
[0078] reverse transcription PCR:
[0079] Reverse Transcription Kit using PrimeScript TM RT-PCR Kit, purchased from Takara Company, commodity number: AK2701.
[0080] The reverse transcription system is as follows:
[0081]
[0082] 65 ℃, the reaction 5min. Take it out and put it on ice, add samples according to the following system, and carry out cDNA first-strand synthesis.
[0083]
[0084] 30°C for 10 minutes; 42°C for 1 hour; 72°C for 5 minutes.
[0085] Nested PCR: (purchased from TAKATA company, item number: 6210A)
[0086] The first round of PCR:
[0087] The reaction system is as follows:
[0088]
[0089]
[0090] The reaction procedure is as follows:
[0091]
[0092] Second round of PCR:
[0093] The reaction system is as follows:
[0094]
[0095] The reaction procedure is as follows:
[0096]
[0097] Nested PCR primer sequences are as follows (5'-3'):
[0098] GSP-RT: CGCCATCAATRTACCAGTTGA (SEQ ID NO: 23)
[0099] LP-leader: GTGGTCCTGGCTGCTCTW (SEQ ID NO: 24)
[0100] F: CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC (SEQ ID NO: 26)
[0101] R: CATGCCATGACTCGCGGCCGGCCTGGCCATGGGGGTCTTCGC TGTGGTGCG (SEQ ID NO: 25)
[0102] Among them, R represents the base A/G, W represents the base A/T, and K represents the base G/T.

Example Embodiment

[0103] Example 2. Screening of SARS-CoV-2 Nanobodies
[0104] The first well of a 96-well microtiter plate was coated with the S protein antigen of the original strain of SARS-CoV-2 at a concentration of 1ug/mL, overnight at 4°C; the next day, the coating solution was poured out and washed with PBST for 3 The first and second wells of the ELISA plate were blocked with BSA and incubated at room temperature for 2 hours; the blocking solution was poured out and washed 3 times with PBST; the phage nanobody library obtained in Example 1 was added to the first well and reacted for 2 hours ; Pour out the liquid, pat dry on clean absorbent paper, and wash 5 times with PBST; add 100 μL of SARS-CoV-2 virus original strain S1 protein to the first well, and react for 1 h; aspirate out the first well The liquid was added to the second well, reacted for 1 h, and the phage bound to BSA was removed; the eluate was collected, and 5 μL was used for titer determination, and the rest was used for amplification.
[0105] The phage eluate was added to fresh Escherichia coli ER2738 bacterial solution (preserved in the laboratory, also commercially available, such as purchased from NEB company), 37 ° C, and let stand for 15 min; add carbenicillin and SB medium, 37 ° C, 220rpm, cultured for 2h; add helper phage M13KO7 (multiplicity of infection MOI=20:1) (purchased from NEB company, product number: N0315S) and kanamycin, culture overnight; the next day, centrifuge to get supernatant, add PEG-NaCl solution Precipitate purified phage.
[0106] The amplified product was screened in the next round to ensure that the addition amount of each round of screening was the same, the antigen coating concentration and the S protein competitive elution concentration were decreased by 2 times, the titer of each round was calculated, and a single clone was picked for amplification and purification. ELISA identification. Positive single clones were obtained after 3 rounds of panning.

Example Embodiment

[0107] Example 3. Expression of SARS-CoV-2 Nanobodies
[0108] The positive monoclonal plasmid was extracted, transformed into E. coli TOP10F' competent cells (purchased from ThermoFishier), and then spread on solid medium for overnight culture after recovery. The next day, a single clone was picked and cultured in SB-carboxybenzyl medium, and IPTG was added to induce overnight expression; the next day, the cells were lysed with a high-pressure homogenizer, filtered through a membrane, and purified with a nickel column, that is, using a histidine tag to bind with the cells. The nanobodies were separated and purified by affinity chromatography of nickel chloride in the nickel column to obtain high-purity anti-SARS-CoV-2 nanobodies, namely antibodies A1-A6. After amino acid sequencing analysis, the amino acid sequences of the obtained nanobodies were as shown in SEQ IDNO: 1-6.

PUM

PropertyMeasurementUnit
Affinity5.15 ~ 25.4nm
Affinity1.27 ~ 10.1nm

Description & Claims & Application Information

We can also present the details of the Description, Claims and Application information to help users get a comprehensive understanding of the technical details of the patent, such as background art, summary of invention, brief description of drawings, description of embodiments, and other original content. On the other hand, users can also determine the specific scope of protection of the technology through the list of claims; as well as understand the changes in the life cycle of the technology with the presentation of the patent timeline. Login to view more.

Similar technology patents

Molding with embedded coupling particles for biomolecules

InactiveUS20100317039A1improve ratiohigh affinity
Owner:FRAUNHOFER GESELLSCHAFT ZUR FOERDERUNG DER ANGEWANDTEN FORSCHUNG EV

Identification of high affinity molecules by limited dilution screening

InactiveUS7754433B2high affinitylow binding affinity
Owner:AMGEN FREMONT INC

Reactive dye compounds

InactiveCN1303415Ahigh affinitySimplified soaping process
Owner:THE PROCTER & GAMBNE CO

Method for dyeing yellow tipping base paper

InactiveCN101967773Ahigh affinityevenly dispersed
Owner:潍坊华港包装材料有限公司

Single azo based pyridine ketone dye and preparation method and application thereof

InactiveCN105504858Ahigh affinityNot easy to change color
Owner:HANGZHOU FLARIANT SPECIALTY

Classification and recommendation of technical efficacy words

  • high sensitivity
  • high affinity

System and method for optical coherence imaging

ActiveUS20060055936A1sufficient signal to noise ratiohigh sensitivity
Owner:THE GENERAL HOSPITAL CORP

Three-axis integrated MEMS accelerometer

InactiveUS20060272413A1low costhigh sensitivity
Owner:VAGANOV VLADIMIR +1

Patterning process, resist composition, and acetal compound

InactiveUS20110236826A1high dissolution contrasthigh sensitivity
Owner:SHIN ETSU CHEM CO LTD

Soluble tgf-b type III receptor fusion proteins

InactiveUS20070184052A1increase activin signalhigh affinity
Owner:THE GENERAL HOSPITAL CORP

Non-mammalian GnRH analogs and uses thereof in the immune system

InactiveUS20050043245A1high affinitygreat affinity
Owner:SILER KHODR THERESA

Method for assaying protein-protein interaction

ActiveUS20050100934A1increase interactionhigh affinity
Owner:LIFE TECH CORP +1

Anti-TNF chimeric antibody fragments

InactiveUS20060140946A1inhibit biological activityhigh affinity
Owner:NEW YORK UNIVERSITY +1
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products