A Protease Activated Biosensing Method For The Detection Of HCV NS3/4A Activity

Hepatitis C virus(HCV)is an RNA virus that is prone to mutation.It is the main cause of chronic hepatitis,cirrhosis and hepatocellular ca rcinoma.More than 3 million people are infected by HCV each year.Currently,there are170-180 million infected people in the world,which is about 3%of the global population.Due to the lack of available animal models,clinically validated vaccines have not been developed.HCV NS3/4A serine protease is a heterodimeric metalloprotease for the hydrolysis and maturation of HCV NS3-NS5B,a non-structural protein of HCV,and plays an important role during HCV proliferation and replication.As a result,NS3/4 A serine protease becomes the target for the treatment of HCV infection,and the related inhibitors were screened as anti-HCV drugs.Detection of NS3/4A protease activity is of great significance for the inhibitor screening and drug development.Most of th e traditional methods for detection of HCV protease activity,such as enzyme-linked immunosorbent assay,chemiluminescence and electrochemical detection method,are time-consuming,and require large and expensive instruents and professional operators.Ther efore,it is urgent to develop new methods,which can detect the NS3/4A protease activity conveniently and rapidly.Lateral flow chromatographic test strip ha s unique advantages,including portability and short detection time.However,the signal visualiza tion relies on the aggregation of chromogenic agents and lacks excellent signal amplification mechanism,which results in low sensitivity of the test strips.Previously,our group has developed a protease-targeted transcription mechanism that activates T7 RNA polymerase for signal conversion and amplification.Here,the signal amplification mechanism was combined with the lateral flow chromatographic test strip to realize the convenient and visual detection of NS3/4A protease activity.This paper has carrie d out the following work:1?Clone and expression of the NS3/4A protease-activated T7 RNA polymerase(recombinant protein PR _NS3/4A)and exploration of the basic properties.The recombinant plasmid p ET28a-pr NS3/4A was constructed based on the molecular cloni ng technology,and the recombinant protein PR _NS3/4A was heterogeneous expressed,where T7 lysozyme was connected to T7 polymerase with the substrate peptide of NS3/4A protease.NS3/4A protease was also expressed.In the presence of NS3/4A protease,PR _NS3/4A can be hydrolyzed into2 pieces,and T7 RNA polymerase is activated.Further,the transcript ion products can combine Th T and produce fluorescence at 495 nm.Hence,the signal conversion and amplification function of PR _NS3/4A was verified,which laid a foundation for the subsequent visual detection of NS3/4A protease activity based on lateral flow chromatographic test strip.2?An lateral flow chromatographic test strip was constructed in response to HCV NS3/4A protease.Combining test strip with the recomb inant protein PR_NS3/4A,we developed a rapid and convenient test strip for the detection of NS3/4A activity.Through the interaction between Au and-SH,the surface of gold nanoparticles was modified with oligonucleotide 1,oligonucleotide 2 and 3were fixed at the text line(TL)and control line(CL)of the test strip,respectively,and oligonucleotide 3 is complementary to oligonucleotide 1.The transcription products of PR _NS3/4A were designed as the sequence complementary to both oligonucleotide 1 and ol igonucleotide 3.In the present of NS3/4A,T7RNA polymerase is activated and initiate to transcribe the RNA,which can form a"sandwich"structure with oligonucleotide 1 and oligonucleotide 2 at TL and capture the gold nanoparticles.The more the gold nan oparticles are captured,the more obvious red color at TL is visible to naked eye,which makes it possible to detect the activity of NS3/4A conveniently and intuitively.After optimization of the concentration of PR _NS3/4A,sensitive and visual detection of HCV NS3/4A protease activity was realized,with the detection limit as low as 5 p M and good selectivity.3?Improvement of T7 RNA polymerase transcription efficiency based on intramolecular interaction.Recognition of the promoter sequence of the template was necessary for T7 RNA polymerase to start the subsequent transcription.In order to shorten the time of T7 RNA polymerase search the promoter and improve the efficiency of transcription,we fused the T7 RNA polymerase with DCV,which can specifically co valently connect to nucleic acid.The fusion protein T7 RNAP-DCV covalently binds to the transcription template to form a transcription complex,which is expected to convert the intermolecular interaction between T7 RNA polymerase and the transcriptional t emplate into intramolecular interaction.The preliminary results showed that this design can improve the transcription efficiency of T7 RNA polymerase by 6 times.