Development Of A Specific Diagnosis Of Sars-COV-2 Spike Protein

The SARS-CoV-2 virus has caused huge social and economic burden since the first report in Wuhan,2019,caused extensive attention at the all over the world.Now,the personnel and cargo transportation are closely related to the epidemic.As a result,we urgently need to develop a fast,accurate,high sensitivity,specificity and operational simplicity testing technology,especially for the point-of-care testing,that is,the existence of living viruse.It has a very important significance for domestic epidemic prevention and cargo exchange between countries.Currently,There is no report,which detects living SARS-CoV-2 virus and its kit or any application thereof that can reach above the requirements.This approach utilized the SARS-CoV-2virus’ s spike protein,which the receptor is angiotensin converting enzyme 2 protein(hACE2)in human body.The main work content was designed for detecting methods for living SARS-CoV-2 virus’ s spike protein,two approaches are as follows:1.Novel SARS-CoV-2 virus’ s S protein was specifically identified by bimolecular fluorescence complementation(BiFC)with recognition peptideUsing the BiFC effect of red fluorescent protein of mCherry and the specific sensitive recognition peptide derived from hACE2 receptor and S protein from SARSCOV-2 virus,the mCherry red fluorescent protein was separated in its protein residue from 159 to 160 and the two segments were respectively connected to the recognition polypeptide to design the DNA sequence coding for two protein fluorescent probes.The DNA sequences of two fluorescent probes were synthesized by a series of molecular experiments,and the expression vectors of two fluorescent probes were constructed.Two protein probes were obtained by prokaryotic expression and purifided by His-tag labeled chromatographic column.The BiFC effect of the two fluorescent protein was restored when two recognition peptides binding to S protein,by which the two fluorescent protein fragments forms the complex and emits the red fluorescence.Consquently,SARS-CoV-2 virus can be detected.It was confirmed that the two fluorescent protein fragments could be triggered BiFC reaction by adding S protein.The BiFC effect of protein fluorescence probe interaction with S protein was measured.2.Novel SARS-CoV-2 virus detection using specific peptide combined with the aggregation-induced emission fluorogen.Using the specific peptide binding to S protein of SARS-COV-2 derived from hACE2 receptor,a fluorescent probe was developed for specific detecting S protein of SARS-CoV-2 using the TPE fluorogen with AIE effect.By chemical synthesis,a fluorescent probe using TPE as fluorogen was successfully synthesized.The experiment of interaction between S protein and probe proved that our detection method has high sensitivity to S protein,and the reaction is stable,and the fluorescence was not apparently decayed in 48 hours.The experimental results showed that the method was not affected by the the proteins and saliva from normal persons.The probe could also interact with adenovirus(SARS-COV-2 pseudovirus)expressing the S protein of SARS-COV-2 virus in different concentrations at room temperature in a short period of time,and could stimulate AIE effect of the probe,producing a strong emission at the peak of 465 nm,which was verified by the above experiment using S protein.The intensity of AIE effect of probe was proportional to the concentration of lentivirus.It is anticipated that this detection method has good application potential.