Novel coronavirus delta mutant strain or prototype strain detection kit and its application
A detection kit, coronavirus technology, applied in the field of biomedicine, can solve problems such as long time
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Embodiment 1
[0039] Example 1: Acquisition of broad-spectrum binding capacity IMCAS-72 and IMCAS-364 antibodies
[0040] Extraction of RNA from peripheral blood lymphocytes of recovered patients and establishment of human single-chain antibody phage display library
[0041] With the informed consent of 12 people who were infected with the new coronavirus and recovered and discharged, 3-10 mL of blood was collected from each, PBMCs were separated, and transferred to 1.5 mL EP tubes. Add 700ml of trizol and leave it for 5 minutes. In the above EP tube, add 0.14ml of chloroform, cover the EP tube, shake vigorously for 15 seconds, stand at room temperature for 3 minutes, and centrifuge at 12000g (4°C) for 15 minutes. Take the upper aqueous phase and place it in a new EP tube, add 0.5 ml of isopropanol, stand at room temperature for 10 minutes, and centrifuge at 12,000g (4°C) for 10 minutes. Discard the supernatant, add 1 ml of 75% ethanol for washing, vortex to mix, centrifuge at 7500g (4°C) ...
Embodiment 2
[0060] Example 2. Surface plasmon resonance technology to detect protein and antibody affinity (biacore 8k)
[0061] The detection of intermolecular interactions using surface plasmon resonance was performed on the Biacore 8K, a biomacromolecular interaction analysis system produced by GE Healthcare. Use biotin-streptavidin coupling method (SA chip) to capture Prototype RBD, Alpha variant RBD, Beta variant RBD, Delta variant RBD, Lamdavariant RBD, Omicron variant RBD protein as stationary phase, mobile phase is IMCAS to be detected -364 and IMCAS-72 COVID-19 neutralizing antibody proteins, and then the kinetic parameters were analyzed and plotted by BIA evaluation software.
[0062] Experimental steps: Using the coupling effect of biotin-streptavidin, we firstly used Prototype RBD, Alpha variant RBD, Beta variant RBD, Delta variant RBD, Lamda variant RBD, Omicron variant RBD proteins with biotinylation reagents in proportion After 30 minutes at room temperature, the protein w...
Embodiment 3
[0066] Example 3. Construction, Expression and Purification of Antibody IgG Whole Antibody
[0067] Antibody IgG Whole Antibody Construction
[0068] To obtain human antibodies for subsequent evaluation, a full anti-IgG1 construct was designed. The strategy is as follows:
[0069] Heavy chain H: CMV promoter-EcoRI-signal peptide (SP)-heavy chain variable region (VH)-heavy chain constant region (CH)-Xhol;
[0070] Light chain kappa: CMV promoter-EcoRI-signal peptide (SP)-light chain variable region (VK)-light chain constant region (CLκ)-Xhol;
[0071] The light and heavy chain variable region sequences were respectively connected with the corresponding expression vector pCAGGS containing the constant regions of heavy chain CH and light chain CLκ through homologous recombination, and cloned into the expression vector pCAGGS to obtain light and heavy chain codes containing specific antibodies. The recombinant plasmid of the gene; wherein, the light and heavy chain variable reg...
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