Research On Hepatitis C Virus With The Beacon Aptamer Targeting For HCV Envelope Protein

Nearly3%of the world population are infected with hepatitis C virus (HCV). Ithas emerged as one of the major pathogens of liver diseases, resulting in acute andchronic infections, of which the chronic hepatitis C can evolve to cirrhosis andhepatocellular carcinoma, and nearly35,000people have been infected each year. HCVis a blood-borne virus, and its early diagnosis is very difficult, so some patients couldeasily miss the best time for treatment because of failure to timely diagnosis.Nowadays, effective HCV vaccine is not available, and the clinical detection methodsare time-consuming, which is prone to misdiagnosis or false positive results. Therefore,current methods barely meet the requirement for high throughput detection and rapidblood screening, and improved novel methods of early detection are badly demanded.Envelope glycoproteins E1and E2are two structural proteins of HCV viralparticle, and they are glycosylated to form a non-covalent complex-E1E2, which playsa key role in virus infection and virus particle formation, by participating in theinteraction between the cell and the receptor and inducing the production ofneutralizing antibodies. These have made HCV envelope glycoproteins a favoredresearch subject.We firstly cloned HCV E1E2gene to pET28b vector, and the E1E2protein hadbeen expressed and purifed in large amount. Then we performed an ssDNA screeningto obtain aptamers, which can specifically bind to E1E2, by systematic evolution ofligands by exponential enrichment(SELEX). To study the binding function of theseaptamers, we expressed and purifed E1and E2protein separately, and theenzyme-linked oligonucleotide assay (ELONA) showed that these aptamersspecifically bind to E2rather than E1. Lastly, we modified aptamer into a molecularbeacon aptamer (MBA), which acting as a signal probe, and we were able to detect theE1E2and E2protein from a nM level, as well as detecting the envelope protein ofHCV viral partical from the supernatant of virus-infected cells.These results indicated that MBA can be used to study the function of HCVenvelope protein in HCV lifecycle. Also, MBA can be potentially applied to the early detection of HCV, thus be developed into a new method for early diagnosis of HCV.