Detection Method Of Hepatitis C Virus Core Antigen Based On Aptamer Techology

Hepatitis C is an infectious disease caused by hepatitis C virus (HCV),which is spread primarily by blood-to-blood. Nearly3%of the populationsworldwide areinfected by HCV. HCV infection often leads to serious liver diseasesincluding chronic hepatitis, cirrhosis and hepatocellular carcinoma. Currently, there isno vaccine in the world. The HCV genome is a single-stranded RNA of positivepolarity which encodes a long polyprotein of about3010residues. HCV core proteincan lead to malignant transformation of hepatocytes, so as to form the malignanttumors by affecting cell cycle, mediating apoptosis, affecting the chromosome injury,gene mutations, transcription factors, mitochondria and other links. HCV core proteinwill appear in the blood in one week after HCV infection, it is one of the the mainmarkers of HCV infection on early stage, becoming an important detect index of HCVinfection.There are many disadvantages of the existing detection method of anti-HCVantibody, such as the window period is long, and the detection reagent quality is notstable etc. HCV RNA detection by PCR also exist a certain limitation because of thedetection method with complex operation, expensive instruments and easy to crosscontamination, so it is not suitable for blood or blood product screening, also hard topromote the use in basic-level hospitals. Therefore, it is important to explore anddevelop new diagnosis method for HCV infection early.The nucleic acid aptamers are single-strand oligonucleotides with highspecificity and affinity for their targets, which are screened from a large syntheticDNA/RNA pool by systematic evolution of ligands by exponential enrichment(SELEX) technology. They are characteristic of high affinity and specificity, easilysynthesized and modified in vitro.We inserted HCV core genetic fragments into the vector pLM1to construct theHCV core protein expression recombinant plasmid pLM1-APJFH1-core by genecloning techniques. We obtained a large amount of target protein after inducedexpression and purified the protein using Ni-NTA purification column. We obtainedspecifc aptamers binding HCV core protein from a pool after6rounds of selection bySELEX technology, and we characterized their specificity by fluorescent signaldetection method. We built a new technology-ELONA (Enzyme-linked oligonucleotide assay) based on selected aptamers. We detected the recombinant HCVcore protein and that in clinical patients’ serum infected by HCV. ELONA canimprove the detection sensitivity, shorten the window period, and it may realize theearly diagnosis of HCV and clinical screening in general population, then providesguidance for other disease in the early diagnosis and clinical screening.