Study On The Broad-spectrum Inhibition Of Coronaviruses By Antisense RNAs And Its Sensitization Effect By Bioactive Peptides

Objective: Coronavirus and its variants that cause COVID-19,SARS,MERS,etc.,seriously infringe human life,health and economic and social development.There is an urgent need to develop broad-spectrum anti coronavirus drugs,effectively resist COVID-19 and its variants,and treat serious diseases caused by known and newborn coronaviruses.At present,there are no reports on conserved antisense RNAs that can broad-spectrumly inhibit coronavirus at home and abroad.This paper explores the broad-spectrum inhibitory effect of antisense RNA on coronavirus and the sensitization of bioactive peptides.Methods:(1)Fifty conserved regions and conserved sequence were explored by whole genome supercomputing analysis and RDRP gene alignment of coronavirus.(2)Construct RDRP-EGFP fusion gene expression plasmids for eight types of coronavirus infections in humans.(3)Cell culture and Lentivirus infection and transfection of RDRP coronavirus conserved region gene,antisense RNA(5-1)expression vector,antisense RNA(6-1)expression vector.(4)Integrate 5 antisense RNA sequences into a single expression vector for tandem expression,and integrate 6 antisense RNA sequences into a single expression vector for tandem expression.Use fluorescence quantitative RT-QPCR to detect the inhibitory effects of antisense RNA(5-1)and antisense RNA(6-1)on the expression of RDRP genes in eight types of coronavirus infections,as well as the inhibitory effects on the expression of RDRP proteins in eight types of coronavirus infections.(5)293T cells were cultured in vitro,transfected with conventional cells,and analyzed by flow cytometry.Result:Compared to the negative control(SH1),the results of flow cytometry and real-time fluorescence quantitative RT-QPCR analysis showed that:(1)the tandem expression of five antisense RNAs(5-1)inhibited the expression of RDRP protein and RNA in eight types of coronavirus infections,reaching 52.96%-76.41% and 0.00%-98.57%,respectively;(2)Six antisense RNAs were expressed in tandem(6-1),which effectively inhibited the protein and RNA expression of eight types of coronavirus RDRP infecting humans,reaching 53.45%-76.53%and 0.00%-87.91%,respectively;(3)Five antisense RNAs were expressed in tandem(5-1),and 20 uL of bioactive peptides were added 30 minutes before transfection,which on average inhibited 46.78% of the protein expression of the 2019 nCoV coronavirus RDRP.(4)The analysis of RT-QPCR results showed that five antisense RNAs were expressed in tandem(5-1),and adding 20 uL of bioactive peptides 30 minutes before transfection could inhibit an average of 87.39% of the RNA expression of the 2019 nCoV coronavirus RDRP.Conclusion:(1)Five conserved antisense RNAs expressed in tandem and six conserved antisense RNAs expressed in tandem can significantly and efficiently inhibit COVID-19 and its variants,significantly and efficiently inhibit all eight known coronaviruses infecting humans in a broad spectrum,which is of great theoretical and practical significance for the treatment of diseases caused by coronavirus and its variants and newly born highly pathogenic coronavirus,and can be used to develop broad-spectrum anti coronavirus drugs,It has good application prospects.(2)Bioactive peptides can enhance the sensitivity of antisense RNA against COVID-19 RDRP,which is of great value for further research.