Recombinant receptor binding protein and recombinant receptor protein for detecting new coronavirus neutralizing antibody
A receptor binding and receptor protein technology, applied in the field of biomedicine, can solve problems such as insufficient binding stability, false negatives, and restrictions on the continuous detection of neutralizing antibodies against the new coronavirus, and meet the needs of tracking and detection of neutralizing antibodies. The effect of simple operation and low detection cost
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Embodiment 1
[0266] Example 1: Plasmid construction and protein expression and purification of recombinant ACE2 monomeric protein
[0267] 1. Experimental method and steps
[0268] 1.1 Design and construction of expression plasmid: pCDNA3.1(+)-SecreconAAA-ACE2-His plasmid expresses a fusion protein, which contains the pepidase domain (pepidase domain, amino acid 19-615) of ACE2 protein and C Terminal His tag. The engineered Secrecon signal peptide was used, and three alanine sequences were added (SecreconAAA, reference https: / / doi.org / 10.1371 / journal.pone.0155340). The synthesis of DNA fragments and gene cloning were commissioned by Suzhou Jinweizhi Biotechnology Co., Ltd.
[0269] 1.2 Protein expression and purification
[0270] 1.2.1 pCDNA3.1(+)-SecreconAAA-ACE2-His plasmid extraction
[0271] Use the endotoxin-free plasmid large-scale extraction kit to extract the plasmid according to the steps. After measuring the OD value, store at -20°C.
[0272] 1.2.2 Expi 293F cell culture an...
Embodiment 2
[0293] Example 2: Plasmid construction and protein expression and purification of recombinant ACE2-FFc protein
[0294] 1. Experimental method and steps
[0295] 1.1 Design and construction of expression plasmid: pCDNA3.1(+)-SecreconAAA-ACE2-FFc plasmid expresses a fusion protein, which contains the pepidase domain (pepidase domain, amino acid 19-615) of ACE2 protein and Fc tag, and the ACE2 protein extracellular region and the Fc tag are connected through a flexible linker (Flexible linker) (reference doi:10.1016 / j.addr.2012.09.039.). The engineered Secrecon signal peptide was used, and three alanine sequences were added (SecreconAAA, reference https: / / doi.org / 10.1371 / journal.pone.0155340). The synthesis of DNA fragments and gene cloning were commissioned by Suzhou Jinweizhi Biotechnology Co., Ltd.
[0296] 1.2 Protein expression and purification
[0297] 1.2.1 pCDNA3.1(+)-SecreconAAA-ACE2-FFc plasmid extraction
[0298] Use the endotoxin-free plasmid large-scale extracti...
Embodiment 3
[0323] Embodiment 3: Expression plasmid construction and protein expression purification of ACE2-RFc protein
[0324] 1. Experimental method and steps
[0325] 1.1 Design and construction of expression plasmid: pCDNA3.1(+)-SecreconAAA-ACE2-RFc plasmid expresses a fusion protein, which contains the pepidase domain (pepidase domain, amino acid 19-615) of ACE2 protein and Fc tag, and the extracellular region of ACE2 protein and the Fc tag are connected by a rigid linker (reference doi:10.1016 / j.addr.2012.09.039.). The engineered Secrecon signal peptide was used, and three alanine sequences were added (SecreconAAA, reference https: / / doi.org / 10.1371 / journal.pone.0155340). The synthesis of DNA fragments and gene cloning were commissioned by Suzhou Jinweizhi Biotechnology Co., Ltd.
[0326] 1.2 Protein expression of ACE2-RFc
[0327] 1.2.1 pCDNA3.1(+)-SecreconAAA-ACE2-RFc plasmid extraction
[0328] Use the endotoxin-free plasmid large-scale extraction kit to extract the plasmid ...
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