A kind of antibody that specifically binds novel coronavirus s protein and its application
A specific and protein technology, applied in the direction of antiviral immunoglobulin, virus/bacteriophage, virus, etc.
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[0049] Preparation of monolayer Vero cells: resuscitate Vero cells (purchased from ATCC, USA) into T25 cell flasks filled with DMEM medium (purchased from Gibco), culture at 37°C for 24 hours, discard the old medium and replace with new ones. Continue to culture at 37°C for 7 days to obtain a monolayer of Vero cells; use DMEM medium to expand the monolayer of Vero cells at a ratio of 1:6 until they are enlarged from the cell flask to 10-layer cell factory, the cell factory is filled with a single layer of Vero cells and used for inoculation of the new coronavirus;
[0050] Preparation of SARS-CoV-2 virus liquid: Take Vero cells cultured into a dense monolayer, discard the cell supernatant, and wash 3 times with PBS buffer; inoculate the new coronavirus (isolated by Zhejiang Provincial Center for Disease Control and Prevention, number ZJ12) at 0.001 MOI Inoculated in a single layer of Vero cells, cultured at 37°C for 60 minutes, added virus maintenance solution (MEM medium, pur...
Embodiment 1
[0068] Example 1: Preparation, purification and identification of SARS-CoV-2 S protein specific antibody
[0069] 1.1 Polypeptide synthesis and animal immunity
[0070] The S protein of SARS-CoV-2 was analyzed by means of bioinformatics, and the two peptides 393-448aa and 449-521aa in the receptor binding domain (RBD) region were selected and marked as R1 and R2 respectively. The amino acid sequence is shown in Table 1.
[0071] Through in vitro synthesis, two polypeptides R1 and R2 are obtained, and hemocyanin is coupled to the N-terminus by a cross-linking agent. The in vitro synthesis and coupling were completed by Nanjing GenScript Company.
[0072]R1 and R2 coupled with hemocyanin were used to immunize New Zealand rabbits (female, weighing 2-3kg, purchased from Shanghai Jiesijie Company). The specific immunization procedure was as follows: three times of immunization were carried out on day 0, day 14 and day 21 respectively, blood was collected and serum was separated ...
Embodiment 2
[0085] Example 2: Application of SARS-CoV-2 S protein-specific antibody
[0086] 1. Establishment of detection method for SARS-CoV-2 S protein
[0087] 1.1 Preparation of SARS-CoV-2 Standards
[0088] In order to ensure that the test results can be quantified, SARS-CoV-2 standards are prepared first.
[0089] The preparation method of SARS-CoV-2 standard substance is as follows: take virus titer as 6.5 lgCCID 50 / mL of SARS-CoV-2 virus harvesting liquid 5 L, add 1.25 mL of BPL (β-propiolactone, purchased from SERVA company) at 1:4000 (BPL: virus harvesting liquid = 1:4000, v / v) Inactivate at 4°C for 24 h, hydrolyze at 37°C for 2 h, and then clarify with a continuous-flow centrifuge with a clarification parameter of 700 mL / min and a centrifugal force of 8,000 g to obtain a clarified virus; The solution was concentrated to 1 / 100 of the original volume, and the virus was purified by sucrose density gradient ultracentrifugation. Centrifuge at 30,000 rpm for 2 h; after centrifu...
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