The Rapid Selection Of HIV-P24 Antigen Aptamers By Agar Beads

Human immunodeficiency virus is the cause of AIDS. So far, there is no safe and effective treatment, the main way is prevent universal HIV antigen, antibody and virus detection, in order to discover the infected person as early as possible, to intervent and to prevent the spread of AIDS.HIV-P24 antigen is the core protein of HIV, which plays a very important role in the process of packaging and maturation of the virus. First time, when human were infected with HIV, HIV antigen appeared in the patient's serum earliest and it can be detected after 2 or 3weeks. HIV-P24 antigen is the earliest immune marker, which can be detected in the serum.Early and accurate detection of HIV is helpful for early diagnosis, which can shorten the“window period”. At the same time, P24 detection can predict the course of disease, and the detection is an effective means to track the HIV process. Therefore, it is very necessary to establish a fast and accurate method for detecting P24 antigen.Aptamer is an oligonucleotide fragment by artificial screening in vitro, because of its special two stage structure and three stage structure, which can bind with all kinds of targets such as Antigen, antibody, virus, metal ion, et al. The binding of them is similar to the binding of antigen- antibodies, so aptamers are known as "chemical antibodies". But its some features are better than antibodies: high affinity and specificity, more easily prepared and chemically modified, wide range of targets, small molecules and non-immunogenicity. Therefore,successfully screened out the HIV-P24 antigen specific ligands is expected to establish a rapid,accurate and efficient method for the detection of HIV-P24 antigen, shorten the window period of detection and lay the foundation for the rapid and accurate diagnosis of AIDS.Carboxylated agar beads used as the medium of target binding and HIV-P24 antigen as target molecule to screen the specific oligonucleotide ligands of HIV-P24 antigens by subtractive SELEX technique. In the late stage, the secondary library was transferred into E.coli DH5 and positive clones were selected to detect by interleaved PCR, at last to sequen them.In the whole process of HIV-P24 antigen specific oligonucleotide screening, we changed some factors, such as the amount of beads, lotio, washing times, binding time and the replacement of non-target molecules. Finally, established a set of programs of how to screen such ligands more complete and efficient. Using the subtractive SELEX technology afterseven rounds of screening, the HIV-P24 antigen specific oligonucleotide was successfully screened. The screened aptamers was transformated,cloned and picked clone groups, and the clone groups were identificated through overlapping PCR, and then detect the specificity of them, finally, we got 5 clones which have high specificity to HIV-P24 antigens and we also got the sequencing results.The HIV-P24 antigen specific oligonucleotide ligand was screened by subtractive SELEX technique, the aptamer has strong specificity. A rapid, efficient and accurate method for detecting HIV-P24 antigen based on HIV-P24 antigen specific oligonucleotide is expected to be established. It can shorten the “window period” and lay the foundation for the rapid and accurate diagnosis of AIDS.