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CN-115594758-B - Nanometer antibody for resisting novel coronavirus SARS-CoV-2 and its preparation method and use

CN115594758BCN 115594758 BCN115594758 BCN 115594758BCN-115594758-B

Abstract

The invention relates to the field of biotechnology, and discloses a nano antibody targeting a novel coronavirus SARS-CoV-2 receptor binding domain, a preparation method and application thereof. The invention also discloses biological materials, derivative antibodies, detection reagents and the like related to the nano antibodies. The nanobody of the present invention primarily recognizes the receptor binding motif region that binds RBD. The nanobody includes complementarity determining regions CDR1, CDR2, CDR3, and may further include framework regions FR. The nanometer antibody can be combined with SARS-CoV-2 in high efficiency and specificity, the affinity can reach picomolar level, and the nanometer antibody has relatively high neutralization bioactivity, clear recognition and combination mechanism and excellent effect in SARS-CoV-2 detection or SARS-CoV-2 induced diseases.

Inventors

  • Li Dianfan
  • TAN JINGQUAN
  • Dimitri Ravillet

Assignees

  • 南京晶准生物科技有限公司

Dates

Publication Date
20260505
Application Date
20210628

Claims (11)

  1. 1. A nano antibody for resisting SARS-CoV-2 of novel coronavirus is characterized by that the amino acid sequence of said nano antibody includes complementarity determining regions CDR1, CDR2 and CDR3, and the CDR1 is shown as SEQ ID NO.2, the CDR2 is shown as SEQ ID NO. 3 and the CDR3 is shown as SEQ ID NO. 4.
  2. 2. The nanobody according to claim 1, wherein the nanobody comprises framework regions FR1, FR2, FR3 and FR4 shown in SEQ ID NO. 5, FR2 shown in SEQ ID NO. 6, FR3 shown in SEQ ID NO. 7 and FR4 shown in SEQ ID NO. 8.
  3. 3. The nanobody according to claim 2, wherein the amino acid sequence of the nanobody is shown in SEQ ID No. 1.
  4. 4. An isolated polynucleotide encoding the nanobody of claim 1.
  5. 5. The polynucleotide according to claim 4, wherein the sequence of the polynucleotide is shown in SEQ ID NO. 9.
  6. 6. A construct, characterized in that, comprising the polynucleotide according to claim 4.
  7. 7. A nanobody expression system comprising a construct or genome according to claim 6 having incorporated therein an exogenous polynucleotide according to claim 4 or 5.
  8. 8. A pharmaceutical composition comprising a nanobody according to any of claims 1-3, and a pharmaceutically acceptable carrier.
  9. 9. A detection reagent or a detection kit, characterized in that it contains the nanobody according to any one of claims 1 to 3.
  10. 10. Use of a nanobody according to any one of claims 1 to 3 or a polynucleotide according to claim 4 or 5 or a construct according to claim 6 or an expression system according to claim 7 or a pharmaceutical composition according to claim 8 for: I) The application in preparing the medicine for preventing and/or treating the disease caused by the novel coronavirus SARS-Cov-2; II) application in immunological examination and analysis for non-disease diagnosis and treatment purpose or in preparation of detection reagent or kit for novel coronavirus SARS-Cov-2.
  11. 11. A method for producing a nanobody according to any one of claims 1 to 3, comprising the step of culturing the nanobody expression system under a condition suitable for expression of the nanobody, thereby expressing the nanobody, and purifying and separating the nanobody.

Description

Nanometer antibody for resisting novel coronavirus SARS-CoV-2 and its preparation method and use Technical Field The invention relates to the field of biotechnology, in particular to a nano antibody for resisting novel coronavirus SARS-CoV-2, a preparation method and application thereof. Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belongs to the beta coronavirus and is the causative agent of a novel coronapneumonic disease (COVID-19) found at the end of 2019 and causing global spread. Because of the characteristics of strong infectivity, high transmission speed, higher mortality rate and the like, and no completely effective treatment means, the virus has become one of important infectious diseases which endanger global public health safety, so the drug development aiming at the virus, the preventive vaccine development and the development of related antibodies are particularly urgent. At present, vaccination is a main mode for preventing and controlling infectious diseases, and a plurality of vaccines aiming at SARS-CoV-2 are marketed worldwide, wherein the vaccines comprise inactivated vaccines, subunit vaccines, viral vector vaccines, nucleic acid vaccines and the like, and the vaccine has long research and development period and high preparation cost. Currently, lower vaccination rates and high frequency of viral variation keep us highly critical. Neutralizing antibodies are a class of antibodies that bind to epitopes of viral particles and reduce viral infectivity by blocking viral infection. The neutralizing antibody has wide sources, can be obtained from the blood plasma of a convalescence person, can also obtain the antibody for recognizing multiple epitopes by immunizing different virus antigen epitopes, has relatively mature preparation process and short research and development period, and is an effective means for preventing the virus transmission and infection by researching and developing the high-titer neutralizing antibody aiming at SARS-CoV-2. The SARS-CoV-2 virus infection host relies on recognition and binding of angiotensin converting enzyme 2 (ACE 2) by the receptor domain (RBD) of its spike protein S to host epithelial cells, followed by fusion with the cell membrane, and the virus completes the infection process. Thus, blocking the binding of the S protein to ACE2 is an effective therapeutic measure for preventing, or cutting off, viral infections. Naturally (camelidae and shark) there is a naturally deleted light chain, an antibody containing only heavy chains (heavy chain antibody). The variable region of the antibody is about 12-15kDa, can recognize the binding antigen, has extremely high affinity, and is the smallest active antigen binding fragment, so the antibody is also called as a nanobody. The nano antibody has the advantages of small molecular weight, strong penetrability, easy expression, easy genetic modification, easy combination of a plurality of epitopes and the like. Thus, such antibodies can be used as candidates for effective neutralizing antibodies, and there is currently no marketed nanobody drug against SARS-CoV-2. Disclosure of Invention In order to achieve the above object, the present invention provides the following technical solutions: in view of the above shortcomings of the prior art, the present invention uses the recombinant expressed SARS-CoV-2 RBD protein in vitro to immunize a llama 4 times, then peripheral blood lymphocytes are isolated and total RNA of the cells is extracted, followed by reverse transcription into cDNA. And amplifying the nanometer antibody sequence by using the cDNA as a template and using a specific primer to construct a phage library. Then through 3 rounds of biological elimination screening, FSEC interaction screening, pseudo virus neutralization activity verification, high resolution compound crystal analysis and other technical means, the nanometer antibody of the novel coronavirus SARS-CoV-2S protein RBD structural Domain (RBD) is obtained by separation. The invention discloses an amino acid sequence of the nano antibody, a preparation method (figure 1) and application thereof, wherein the nano antibody has strong specific binding capacity (Kd <1x10 -12 M) with novel coronavirus SARS-CoV-2 and clear binding epitope information, and the application of the nano antibody provides a wider means for diagnosis and treatment of the novel coronavirus SARS-CoV-2 and is used for solving the problems in the prior art. The invention provides a nanometer antibody amino acid sequence (SA 4) of a SARS-CoV-2S protein RBD structural domain of a novel coronavirus, wherein the nanometer antibody sequence comprises three complementarity determining regions CDR1, CDR2 and CDR3, and the amino acid sequence is as follows: 1) Amino acid sequence of SA 4: QVQLQESGGGLVQPGGSLRLSCAASGSFFEFGTVGWFRQAPGKQRELVSRITGNDHRYYADSVKGRFTISRDNDETTVYLQMDSLKPEDTAIYHCNILEGQRWSNYWGQGTQVTVS(SEQ ID NO:1) 2) CDR1 sequence GSFFEFGT (SEQ ID NO: 2)