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Shark source nano antibody targeting SARS-CoV-2RBD protein as well as preparation method and application of shark source nano antibody

A nanobody, sars-cov-2rbd technology, applied in the biological field, can solve problems such as no, and achieve the effects of small molecular weight, high expression, and low cost

Pending Publication Date: 2022-04-22
JIMEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is currently no patent for shark-derived nanobody sequences against SARS-CoV-2 RBD

Method used

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  • Shark source nano antibody targeting SARS-CoV-2RBD protein as well as preparation method and application of shark source nano antibody
  • Shark source nano antibody targeting SARS-CoV-2RBD protein as well as preparation method and application of shark source nano antibody
  • Shark source nano antibody targeting SARS-CoV-2RBD protein as well as preparation method and application of shark source nano antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 SARS-CoV-2 RBD protein immune shark

[0042] Using recombinantly expressed SARS-CoV-2 RBD protein to immunize sharks, the immunization scheme is as follows figure 1 shown. The present invention carries out five immunizations in total, wherein the first three are subcutaneous injections, and the latter two are tail vein injections. The interval between subcutaneous injections was 10 days, and the interval between tail vein injections was 30 days. Thirty days after the third subcutaneous immunization, the tail vein injection was performed. Fifteen days after tail vein injection, shark peripheral blood was collected via tail vein. In the present invention, 3 striped bamboo sharks are used in total, and the RBD protein dose of each shark is 250 μg for each immunity.

Embodiment 2

[0043] Example 2 SARS-CoV-2 RBD Nanobody Screening

[0044] 1) Slowly add the extracted shark blood to the upper layer of an equal volume of 30% (m / v) sucrose solution, centrifuge at 500 g for 20 min, take the middle lymphocyte layer, wash with PBS, and collect the cell pellet by centrifugation. Total RNA was extracted using an RNA extraction kit and reverse transcribed into cDNA.

[0045] 2) Using cDNA as a template and specific primers to amplify the VNAR sequence, the forward primer is: 5'-GCTGCACAGCCTGCTATGGCAACTCAACGGGTTGAACAAACACCGA-3', and the reverse primer is: 5'-GAGTTTTTGTTCGGCTGCTGCTGGTTTTACAGTCAGAATGGTGCCGC-3'. The DNA polymerase used for amplification is a high-fidelity enzyme, and the amplification program is: 98°C, 10s, 57°C, 15s, 72°C, 25s, 30 cycles. The amplified VNAR fragments were recovered using a kit. Using the pR2 phagemid as a template, specific primers were used to amplify the pR2 phagemid. The forward primer was: 5’-AGCAGCCGAACAAAAACTCATCATCTCAGAAGA...

Embodiment 3

[0050] Example 3 Expression and purification of the obtained SARS-CoV-2 RBD nanobody and its Fc fusion protein

[0051] The nanobody sequence in the present invention is constructed into the mammalian expression vector pTT5 with a signal peptide, which is fused with human IgG1 Fc for expression, and the Fc fragment is expressed at the C-terminus, and can be digested with TEV enzyme to obtain the nanobody. The recombinant plasmid was transfected into HEK 293 cells, the culture supernatant was collected, and the Nanobody Fc fusion protein was purified by rProtein A affinity chromatography column. Such as Figure 4 As shown in A, we obtained highly pure SARS-CoV-2 RBD nanobody Fc fusion protein. After digestion by TEV, a highly pure SARS-CoV-2 RBD nanobody protein ( Figure 4 B).

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Abstract

The invention discloses a shark source nano antibody targeting an SARS-CoV-2 RBD protein, a preparation method and application of the shark source nano antibody. Nucleotide sequences of the nano antibody are shown as SEQ ID NO: 1-4, and amino acid sequences of the nano antibody are shown as SEQ ID NO: 5-8. The preparation method comprises the following steps: immunizing mottled bamboo shark with RBD protein, extracting peripheral blood lymphocyte total RNA, and carrying out reverse transcription to obtain cDNA; connecting the VNAR fragment obtained by amplification with the amplified pR2 phages through seamless cloning, and transforming TG1 competent cells to construct a phage antibody library; and screening a nano antibody sequence with SARS-CoV-2 RBD protein specificity from the library, and carrying out recombinant expression to obtain the targeted shark source nano antibody. The nano antibody disclosed by the invention is small in molecular weight, can be used for detecting the SARS-CoV-2 RBD antigen by utilizing the paired nano antibody, and can be applied to immunoblotting of SARS-CoV-2 RBD protein, enzyme-linked immunosorbent assay and COVID-19 pharmaceutical application.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a SARS-CoV-2 antibody and a preparation method thereof, in particular to a shark-derived nanobody targeting the SARS-CoV-2 RBD and a preparation method thereof. Background technique [0002] SARS-CoV-2 belongs to coronavirus, which enters the cell through the receptor binding domain (RBD) of the surface spike protein (spike) and angiotensin-converting enzyme 2 (ACE2) on the surface of epithelial cells, and then enters the cell to complete the infection. [0003] Fully human antibodies isolated from recovered patients have been proven to have good antiviral effects. These traditional monoclonal antibodies are composed of 2 heavy chains and 2 light chains. They have large molecular weight, complex production process, and are not easy to process and transform. limitation. Compared with traditional antibodies, there is an antibody consisting of only 2 heavy chains in camelids or cartilagin...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13C07K16/46G01N33/569G01N33/68A61K39/395A61P31/14
CPCC07K16/10G01N33/56983A61P31/14C07K2317/10C07K2317/569G01N2333/165G01N2469/10A61K2039/505Y02A50/30
Inventor 陈玉磊林锦锦曹敏杰金腾川
Owner JIMEI UNIV
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