Novel coronavirus (sars-cov-2) spike protein binding molecule and application thereof
A SARS-COV-2 and spike protein technology, applied in antiviral immunoglobulin, application, antiviral agents, etc., can solve the problems that it is difficult to become a specific drug for SARS-COV-2, and the treatment effect is not good.
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Embodiment 1
[0075] Screening of single domain antibodies against SARS-COV-2-Spike protein
[0076] 1.1 Library Construction
[0077] 1.1.1 Immunity
[0078] The alpaca was immunized with the Spike-RBD protein of the new coronavirus, and immunized at the 1st, 2nd, 4th, and 6th week, respectively, for a total of 4 times, and each immunization dose was 300ug.
[0079] 1.1.2 Extraction of total RNA
[0080] Get 50ml of peripheral blood of alpaca after the 6th week of immunization, separate lymphocytes, use Trizol to extract the total RNA of lymphocytes, and adopt the ultraviolet spectrophotometer to detect the results of the extracted RNA: OD260 / 280=1.97, OD260 / 230= 2.14, indicating that the extracted RNA is not significantly degraded, and the purity is good; the total RNA concentration is 937.5ng / μL. The extracted total RNA was used for agarose gel electrophoresis, and the results were as follows: figure 1 As shown, two bands of 28S and 18S can be seen.
[0081] 1.1.3 RNA reverse transc...
Embodiment 2
[0142] 1.1 Preparation of Fc fusion protein of SARS-COV-2-Spike protein single domain antibody
[0143] According to the amino acid sequence of the constant region of human immunoglobulin (IgG1) on the protein database Uniprot, the amino acid sequence of the human IgG1-Fc region (SEQ ID NO: 85) was obtained. By reverse transcription PCR, the nucleic acid fragment encoding human IgG1-Fc (nucleic acid sequence such as SEQ ID NO: 107) was obtained from human PBMC total RNA, and then the SARS-COV-2-Spike protein single domain antibody and Fc were obtained by overlapping PCR The nucleic acid fragment encoding the fusion protein was recombined into the vector pCDNA4 (Invitrogen, Cat V86220).
[0144] The successfully constructed pCDNA4 plasmid containing the nucleic acid fragment of the fusion protein of SARS-COV-2-Spike protein single domain antibody and Fc was transfected into HEK293 cells for expression. Specifically, the recombinant expression plasmid was diluted with Freestyle...
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