SARS-CoV-2 virus S protein receptor binding region encoding gene, antibody and application thereof

A receptor binding, sars-cov-2 technology, applied in the field of biomedicine, to achieve the effect of ensuring uniqueness, simple operation and high sensitivity

Active Publication Date: 2021-04-09
HANGZHOU BIOLYNX TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is still a lack of effective neutralizing det

Method used

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  • SARS-CoV-2 virus S protein receptor binding region encoding gene, antibody and application thereof
  • SARS-CoV-2 virus S protein receptor binding region encoding gene, antibody and application thereof
  • SARS-CoV-2 virus S protein receptor binding region encoding gene, antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 SARS-CoV-2 S RBD coding gene optimization and expression

[0069] The amino acid sequence position of the sugar spike protein S RBD (Spike protein RBD, S RBD) of SARS-CoV-2 is aa319-aa541, as shown in UniProt-PODTC2 (SPIKE_SARS2) for details, expressing the SARS-CoV-2 spike protein S The native nucleotide sequence of RBD is as follows (SEQ ID NO: 1).

[0070] AGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAA CTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTA TGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCC TATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTA CTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAA TTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGAT TGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGC TTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCT GTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTT CAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGT TTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGT GTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACA...

Embodiment 2

[0077] Example 2 Expression and purification of SARS-Cov-2 S RBD recombinant protein

[0078] The C-terminus of the expressed protein of SEQ ID NO:2 is linked to the Fc fragment of human IgG1 (P01857) for purification and subsequent detection.

[0079] Synthesize the BRP2 gene fragment, connect the BRP2 gene fragment to the upper pBV vector (Bailing Bio), and transform it into Escherichia coli for 16 hours at 37°C.

[0080] The next day, the BRP2 plasmid was lysed and extracted. According to a conventional method, 50 μg of pBV-BRP2 was used to transfect 50 mL of 293-6E cells with cationic liposomes. After 6 days of transfection, the cells and supernatant were collected, and the collected cells were lysed to prepare a cell lysate. The supernatant after transfection was collected, the recombinant protein was purified using a nickel column, and the buffer was replaced with PBS. Purified samples were stored at 4°C.

Embodiment 3

[0081] Example 3 Identification of SARS-CoV-2 S RBD recombinant protein

[0082] The SARS-CoV-2 S RBD recombinant protein was identified using ELISA, SDS-PAGE, and Western blot respectively. The specific identification methods are as follows:

[0083] 1. Identification of ELISA method

[0084] 1) Coat the ACE2 recombinant protein (with His tag) receptor in the extracellular region (Cat. No.: ACE-HM401, Shanghai Kaijia Biology), dilute the ACE2 recombinant protein concentration in carbonate buffer to 2 μg / mL, 50 μL / well ELISA plate was coated overnight at 4°C.

[0085] 2) The next day, use 0.01M, pH7.4 PBST to place the coated microplate plate on a plate washer and wash the plate 3 times, 250 μL / well.

[0086] 3) Add 100 μL of 3% BSA / PBST solution to each well, block and incubate at 30°C for 1h-2h.

[0087] 4) After washing the plate, add SARS-CoV-2 S RBD recombinant protein ligand starting from 4 μg / mL and 4-fold dilution to each well, 50 μL / well. Incubate at 30°C for abou...

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Abstract

The invention discloses a recombinant protein expressed by an SARS-CoV-2 virus S protein receptor binding region encoding gene, an antibody generated by the recombinant protein and application thereof, and belongs to the field of biomedicine and the field of immunological detection. The encoding gene has a nucleotide sequence as shown in SEQ ID NO: 2. The encoding gene or antibody can be applied to preparation of an antibody for detecting an anti-SARS-CoV-2 virus S protein receptor binding region. The invention discloses an antibody prepared from recombinant protein expressed by the encoding gene and application of the antibody. The encoding gene provided by the invention ensures the uniqueness of protein expression and reduces the difficulty of subsequent purification of the protein. The prepared antibody is high in antigen detection sensitivity and strong in specificity.

Description

technical field [0001] The invention belongs to the fields of biomedicine and immunological detection, and in particular relates to a gene encoding the receptor binding region of the S protein of SARS-CoV-2 virus, an antibody and its application. Background technique [0002] After the new coronavirus vaccine is successfully developed and vaccinated in humans, it is particularly important to effectively identify the new coronavirus and prevent the antiserum detection of the new coronavirus infection. Among them, only antibodies with neutralizing activity can prevent the new coronavirus from interacting with human angiotensin converting enzyme. 2 (angiotensin-converting enzyme 2, ACE2) combination. At present, there are many methods for this type of detection, such as ELISA, kits, and test strips. Immunochromatography test strips are the fastest and most convenient of all tests, and have high specificity and sensitivity without special equipment. And professional technicians...

Claims

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Application Information

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IPC IPC(8): C12N15/50C07K14/165C07K19/00C07K16/10C12N15/85G01N33/68G01N33/569G01N33/577A61K39/42A61P31/14
CPCC07K14/005C07K16/10C12N15/85G01N33/6854G01N33/56983G01N33/577A61P31/14C12N2770/20022C07K2319/30C07K2317/565C07K2317/76C12N2800/107G01N2333/165G01N2469/20G01N2469/10
Inventor 潘丽李明振蔡宁
Owner HANGZHOU BIOLYNX TECH CO LTD
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