Recombinant novel coronavirus COVID-19S-RBD protein and preparation method and application thereof
A technology for COVID-19S-RBD and coronavirus, which is applied in the field of recombinant novel coronavirus COVID-19S-RBD protein and its preparation, can solve the problems affecting the antigenicity of recombinant protein, complex production process, and reduced detection performance, so as to achieve convenient Effective control of the epidemic, high protein expression, and easy operation
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Embodiment 1
[0047] Embodiment 1: Preparation of recombinant novel coronavirus COVID-19 S-RBD protein
[0048] 1. Synthesis of COVID-19 S-RBD fusion protein gene sequence and construction of expression plasmid
[0049] Artificially synthesized S-RBD fusion protein gene sequence (respectively containing endonuclease Nde I and EcoR I restriction sites before and after), its DNA sequence is shown in SEQ ID No.1, and the corresponding amino acid sequence is shown in SEQ ID No.2 Show. The S-RBD gene was cloned into the pET-28a vector, so that 6 histidines were attached to the C-terminus of the protein to facilitate later purification; the correct positive single clone was screened for expression in the E. coli expression system; among them, the size of the S-RBD protein was is 30kDa.
[0050] 2. Recovery of puncture bacteria
[0051] Take a clean pipette tip, insert it along the pale yellow E. coli growth line in the puncture tube, and repeat the operation several times, changing the directi...
Embodiment 2
[0083] Embodiment 2: Preparation of COVID-19 S-RBD protein colloidal gold detection reagent card
[0084] 1. S-RBD protein coated nano colloidal gold
[0085] (1) Coating: take 1ml colloidal gold, add K 2 C0 3 , then add S-RBD protein, and mix with a rotary mixer for 15 minutes; take another 1ml colloidal gold, add K 2 C0 3 , then add the chicken IgY antibody, and mix with a rotary mixer for 15 minutes;
[0086] (2) Sealing: add BSA to seal, and mix with a rotary mixer for 15 minutes; centrifuge at 6000rpm for 10 minutes to take the supernatant, repeat centrifugation for 10 minutes, take the supernatant, add 100 μl pure water to obtain colloidal gold working solution 1 and colloidal gold working solution 2.
[0087]2. Preparation of colloidal gold pad: mix colloidal gold working solution 1 and colloidal gold working solution 2, dilute with diluent (PBS+2% trehalose) to make gold paving dilution, add colloidal gold working solution 1 to a volume of 1 / 8 of the volume of th...
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