The Aptamers Of HBsAg Were Screened By Subtractive SELEX

Objective Aptamers are single-stranded oligonucleotide, which are selected from random single-stranded oligonucleotide libraries synthesized artificially by SELEX technology. Aptamers can recognize and combine with their target molecules of high specificity and high affinity. Aptamers have many advantages as follows. It is easy to be prepared and modified of aptamers. They have high specificity and high affinity with their target molecules.Their molecular weight is low and range of target molecules is wide. Aptamers are stable. Hepatitis B is a potentially life-threatening liver infection caused by the hepatitis B virus (HBV). Hepatitis B surface antigen (HBsAg) is a main sign of HBV infection, also is the earliest serum index. The detection of HBsAg is greatly significant for early diagnosis of hepatitis B. At the early stage of HBV infection, HBsAg concentration in serum is low. It may not be detected by the existing detection reagents and methods, result in false negative and treatment be delayed. To achieve the objective that laid the foundation for the clinical early diagnosis of the hepatitis B by aptamer technology, the aptamers of HBsAg was screened.Methods HBsAg was chosen as the target molecule and epoxy resins was as screening medium in this experiment. Aptaners of HBsAg were screened by subtractive SELEX technology. The subordinated library was identified by protein-nucleic acid dot blot assay and to observe the ability of the subordinated library binded to HBsAg. Then the subordinated library was recombined with pMD18-T Vector and the recombinants were transformed into E.coli DH5a. We obtained the positive clone. All of the positive clone was identified by protein-nucleic acid dot blot assay and finded the ssDNAs which combined with HBsAg with high specificity and high affinity. At last the ssDNAs were sequenced and assayed.Results (1) After16rounds screening, the enriched library to HBsAg was obtained. Protein-nucleic acid dot blot assay indicated that the enriched library binded with HBsAg well.(2) After transformation and clone, using Protein-nucleic acid dot blot assay finded that positive clone H4-1,H4-4and H4-6combined to HBsAg with high specificity and high affinity. The three aptamers’secondary structure was stem-loop structure mainly. Conclusion We screened three aptamers to HBsAg in our study. It was laid a solid foundation for early trace detection of HBsAg that was using aptamers.