SARS-CoV-2 (severe acute respiratory syndrome-corona virus disease-2) inhibitor and application thereof

[0075] The cultivation and material preparation of embodiment 1 virus

[0076] The SARS-CoV-2 virus was isolated by the patent applicant from the peripheral blood of a Jiangsu patient in the acute phase in 2020. After the virus was inoculated into Vero cells, at 37°C, 5% CO 2 Cultured for 5 days under certain conditions, collected the supernatant and determined the 50% tissue infection dose (50% tissue culture infectious dose, TCID 50 ). All manipulations are performed in BSL-3 laboratories.

[0077] Recombinant S protein (SARS-CoV-2-S), commercially purchased, the sequence is shown in SEQ ID NO.1.

[0078] Example 2 Construction of ScFv human antibody library and screening of anti-SARS-CoV-2-S protein single chain antibody

[0079] 2.1 Materials

[0080] Primers: Family-specific light chain (Vκ and Vλ), IgG heavy chain (VH) and overlap-PCR primers were designed according to the book "Phage Display", including Vκ12 pair, Vλ24 pair, VH 6 pair, and overlap-PCR1 pair.

[0081] Primers for constructing human scFv library:

[0082] Vκforward primers:

[0083] 5'GGGCCCAGGCGGCCGAGCTCCAGATGACCCAGTCTCC 3' (SEQ ID NO.66)

[0084] 5'GGGCCCAGGCGGCCGAGCTCGTGATGACYCAGTCTCC 3' (SEQ ID NO. 67)

[0085] 5'GGGCCCAGGCGGCCGAGCTCGTGWTGACRCAGCTCC 3' (SEQ ID NO. 68)

[0086] Vκreverse primers:

[0087] 5'GGAAGATCTAGAGGAACCACCTTTTGATYTCCACCTTGGTCCC 3' (SEQ ID NO. 69)

[0088] 5'GGAAGATCTAGAGGAACCACCTTTTGATCTCCAGCTTGGTCCC 3' (SEQ ID NO. 70)

[0089] 5'GGAAGATCTAGAGGAACCACCTTTAATCTCCAGTCGTGTCCC 3' (SEQ ID NO. 71)

[0090] 5'GGAAGATCTAGAGGAACCACCTTTGATATCCACTTTGGTCCC 3' (SEQ ID NO. 72)

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[0134] Example 3 Candidate scFv monoclonal microneutralization experiment (all operations were carried out in BSL-3 laboratory)

[0135] 3.1 Preparation of phage antibody

[0136] Pick 15 single colonies that were positive in the Phage-ELISA test and place them in 2ml 2×YT medium (containing 100 μg / mL ampicillin, 12.5 μg / mL tetracycline, and 1 g / mL glucose), culture them overnight at 37°C with shaking, and the next day 1:10 were inoculated into 10ml of 2×YT medium (containing 100 μg / mL ampicillin and 30 μg / mL tetracycline) and cultured with shaking at 37°C for 6 hours, and the helper phage VCSM13 (final concentration of 1×10 9 PFU / mL), incubate at 37°C for 1 h, add kanamycin (final concentration is 50 μg / mL), shake at 30°C overnight, centrifuge at 900 g for 30 minutes, discard the precipitate, add 5×PEG / NaCl to the supernatant, mix Put it on ice for 6 hours, centrifuge at 900g for 30min, discard the supernatant, resuspend the pellet with 1ml PBS, centrifuge at 900g for 30min,...