Selection And Application Of DNA Aptamers For HBeAg

Hepatitis B is an infectious disease caused by the hepatitis B virus (HBV) that affects the liver. Roughly 30% of the world's population show serological evidence of current or past infection. And about half the total liver cancer mortality in 2010 was attributed to HBV infection. These data have led WHO to include viral hepatitis in its major public health priorities.Viral protein of clinical importance include the envelope protein, hepatitis B e antigen and hepatitis B core antigen. Hepatitis B e antigen (HBeAg) is a soluble nucleocapsid protein. HBeAg appears in the serum during the high replicative phase of HBV infection, which makes it a serum marker of active viral replication. It is also used to identify highly infectious mothers.The most typical detection method for HBeAg is ELISA (enzyme-linked immunosorbent assay). However, there are several disadvantages of ELISA, such as time consuming, low precision, un-uniformity of detect results, and false positive deviation. Aptamers are single-stranded nucleic acid ligands. Aptamers can be considered as nucleic acid analogues of antibodies. They possess high affinities to target molecules, with dissociation constants in the nanomolar or picomolar range. In this research, we select novel aptamers that recognize and bind to HBeAg with appreciable affinity and selectivity. Later, a gold nanoparticle-based colorimetric aptasensor and fluorescence bioscensor for the qualitative detection of HBeAg are developed.1) Selection and analysis of HBeAg aptamer:Aptamers are generated by an in vitro selection process called SELEX (systematic evolution of ligands by exponential enrichment).With HBeAg modified magnetic beads as targets, a synthetic DNA library is subjected to selection using SELEX technology. By enzyme-linked oligonucleotide sorbent assay, the selection efficiency, affinity and specificity of selected aptamers are investigated. The secondary structures of aptamers are predicted and analyzed by DNAMAN software. (Chapter Two)2) Aptamer-based fluorescence biosensors for the quantitative detection of HBeAg:We present a simple strategy for aptamer-based fluorescence biosensors for the quantitative detection of HBeAg. The best group for binding HBeAg is labeled with fluorescence and then serves as the molecular recognition element. A short DNA molecule that is complementary to the aptamer serves as the competitor. The short DNA molecule is labeled with a quencher. In the absence of HBeAg, competitor DNA bind to aptamers, resulting in fluorescence quenching. However, in the presence of HBeAg, the competitor DNA are partially displaced from aptamers by HBeAg, leading to the restoration of fluorescence. (Chapter Three)3) A gold nanoparticle-based colorimetric aptasensor for the detection of HBeAg:The gold nanoparticles are synthesized with sodium borohydride reduction. We use ultraviolet specrophotometer and transmission electron microscopy to analysis the properties of gold nanoparticles. The aptasensor is based on the unique optical properties of gold nanoparticles. (Chapter Four)...