Exploration Of SARS-CoV-2 Receptor And Spike Protein Synthesis In Cell-free Based Biological Systems

SARS-CoV-2,an epidemic virus that is extremely dangerous to human society,is transmitted by human-to-human transmission and spreads at a very rapid rate in the population,but it is uncertain whether there are other routes of transmission of SARS-CoV-2.There are many reports of infection in dog,cat and tiger species.Cellular angiotensinconverting enzyme 2(ACE2),the receptor for SARS-CoV-2,is similar in many species,ACE2 protein,as a single transmembrane protein,can be overexpressed in cells,causing problems such as incorrect folding of the protein,and the cell-free system is a novel in vitro expression system for the expression of membrane proteins,in which the exogenous addition of the constructed artificial biofilm to the cell-free system also plays a role in the correct folding of membrane proteins.So this experiment selected 22 species-derived ACE2 proteins and used a cell-free system for expression and functional validation,completing a rapid host screening process for potential hosts of SARS-CoV-2.This is a rapid screening process for potential hosts of SARS-CoV-2.At the same time,to face the mutation problem of SARS-CoV-2,the conserved sequences of the SARS-CoV-2echinoderm protein Spike proteins were classified,and the preliminary expression was performed using different expression systems to explore the design of a universal vaccine for SARS-CoV-2 in the early stage of research.The specific experiments are as follows.(1)A variety of Nanodiscs were constructed by self-assembly of ten membrane scaffold proteins with four liposomes.The cell-free expression system of membrane proteins was used to test and screen the effective Nanodiscs,and they were applied to the cell-free expression system of the SARS-CoV-2 receptor protein ACE2.(2)The expression and functionality of the SARS-CoV-2 receptor protein ACE2 truncated and full-length types from different species were verified using a cell-free system,followed by the exogenous addition of Nanodisc during the cell-free expression of the fulllength type of ACE2 protein to help the expression of this single-transmembrane protein in vitro and to compare the expression and functionality of ACE2 protein with different structures.(3)The Spike proteins were classified as conserved sequences,and preliminary expression of the classified sequences was carried out using mammalian cells,Pichia pastoris,Escherichia coli and cell-free systems to select the best expression system for subsequent experiments.The expression and functionality of the novel coronavirus receptor protein ACE2 differed among species origins,with the strongest binding ability in wildlife and very weak binding in aquatic animals;the cell-free system with exogenous addition of nanodiscs changed the expression and functionality of the receptor protein ACE2 for all species origins,but still the strongest binding was in wildlife,and this system could help the correct folding of this membrane protein and This system can help in the correct folding of this membrane protein and help in further characterization of its functionality.This system allows for a rapid screening of potential hosts for novel coronaviruses,making it an excellent screening mechanism.The expression of conserved fragments of novel coronaviruses also provides a basis for universal vaccine studies.