Selection Of HBsAg-specific Aptamer

The aptamer is a non-natural structure, can be folded single-stranded nucleic acid and can capture specific molecules. Aptamer is a nucleic acid molecule selected from a random oligunucleotide library by the Systematic evolution of ligands by exponential enrichment,(SELEX technology), and aptamer has the ability of molecular recognition. Aptamers have promising advantages these include a wide range of target molecules, flexibility, simple and rapid of preparation, can be simple to molecular modification, and can be repeatly denaturation and renaturation et.,al, These properties of aptamers have led to their application in many areas of biomedical sciences such as purification, drug development, disease diagnosis and treatment.Hepatitis B surface antigen (HBsAg), is a membrane protein of hepatitis B virus, it is accompanied by replication of the hepatitis B virus DNA, and the earliest emergence and the highest titer in the HBV infection, it is an important indicator of early diagnosis of hepatitis B. In the clinical, using the Enzyme-linke immunosorbent assay to detect the HBsAg concentration in the serum of an infected person, the minimum detectable concertration is0.2ng/ml, however, the HBsAg concentration in the blood of some HBV infected person is so low that the ELISA method may not be detected, resulting in false negative results of HBsAg. So accurately detect the concentration of HBsAg is essential for HBV early diagnosis.The objective of this study is that to select HBsAg-specific aptamer by the SELEX technology, further molecular modification, to establish HBsAg specific aptamer-based of a PCR detection assay at the molecular level, and to solve the lack of clinical common detection methods.Specific research methods are as follows:(1) To optimize the ssDNA preparation method. We main use the asymmetric PCR method, stem-loop primer and the length of chain to explore it;(2) To measure the experimental materials (HBsAg)’s activity by HBV assay kit;(3) To establish the nitrocellulose membrane-ELISA and the resin-ELISA methods, and to detect the affinity of these two carriers with the target molecules (HBsAg);(4) To use the two carriers for SELEX screening, compare the screening enrichment effective of them. And by agarose gel retardation assay (EMSA) and fluorescence real-time quantitative PCR methods to identify the final library’s activities of the two carrier’s screening.Through the above research, we get some results as follows:1. We use the three different methods to successfully prepared the ssDNA. The asymmetric PCR method has a higher preparation of rate. The stem-loop method and the length of chain method have accurately and quickly find the location of the ssDNA; in the length of chain method, we labeled primer with fluorescence, and can more accurately judge the ssDNA. Compared the three method, we obtain the length of chain method is the best method to prepare the ssDNA, and it lay the foundation for further SELEX screening;2. By the HBV assay kit to detect the activity of experimental material, result show that is positive;3. We successfully established two carrier-ELISA detection methods, and detected the target molecule (HBsAg) can combined with the nitrocellulose membrane and resin carriers, to ensured the accuracy of the following SELEX screening, also enhanced the purpose of screening.4. By compared the PAGE, agarose gel retardation assay and real-time PCR detection results, we found that the enchriment effective of the resin as a carrier for SELEX screening is better to nitrocellulose membrane’s screening. Resin has weak background adsorption, can firmly fix the HBsAg, and enhance the purpose of screening, we successfully established a method of optimize SELEX screening carrier.We finished the13SELEX screening with resin as a carrier, the positive enrichment effective of resin screening is good, lay the foundation for the subsequent transfer plasmid research and dot-blot experiment.