Screening Of Aptamers To HIV-P24

Aptamers are a class of oligonucleotide molecules screened through SELEXtechnique, which can specifically bind target molecules with high affinity and playbiological effect. Aptamers have many biochemical merits, such as wide range oftarget molecules, small molecular weight, low immunogenicity, easily reconstructedand marked, etc. Consequently, aptamers reveal important application value in thefield of clinical diagnosis and therapy of disease. P24protein is core antigen of HIV-1,which can be used as serum marker of detecting HIV infection in window period. Also,in clinical medicine the determination of P24in the blood is an important indicator ofAIDS condition monitoring. In this study aptamers to HIV-P24were screened throughSELEX technique, which might lay the foundation for detecting P24by utilizingaptamer. This is of great significance to the diagnosis and treatment of AIDS.In this experiment, with recombinant HIV-P24for the specific target,oligonucleotides binding to HIV-P24were screened from a random oligonucleotidelibrary through subtractive SELEX. The binding capacity between oligonucleotidesobtained by SELEX technique and HIV-P24was validated via ElectrophoreticMobility Shift Assay (EMSA). The sub-library oligonucleotides binding to P24andpMD18-T vector were constructed recombinant plasmids by restriction enzymedigestion and ligation reaction. Then the recombinant plasmids were transformed intoE.coli JM-109to clone and separate different oligonucleotides. OligonucleotidessDNA was preparated by asymmetric PCR. The aptamers strongly binding toHIV-P24were screened and the recognition specificity of aptamers for HIV-P24wastested by Dot-blot method.After12rounds SELEX screening, oligonucleotide sub-library enrichment forHIV-P24was obtained. Gel Retardation experiment showed that oligonucleotidesobtained by SELEX technique were able to bind HIV-P24.7aptamers with strongbinding capacity to HIV-P24were screened out by Dot-blot. The binding specificityindicated that No.18and No.27apatmers only bond with HIV-P24, not with humanserum albumin, bovine serum albumin and skimmed milk powder.2aptamers specifically recognizing and binding to HIV-P24have been obtained,this study provides an experimental basis for HIV-P24detection by utilizing aptamerof HIV-P24.