Aptamer-based Detection Of Vascular Endothelial Growth Factor165(VEGF₁₆₅) In Vitro

Vascular endothelial growth factor (VEGF) is a kind of important vasculargrowth factor which promotes the division growth of endothelial cells andangiogenesis. VEGF165is the one with most strong promoting ability of promote thegrowth of vascular and plays important role in basic research, signaling pathways,clinical diagnosis, so how quickly and accurately determine its concentration becomethe focus of the study. Protein detection method is commonly used antibody as a tool,Because of its own shortcomings, limiting its application. Aptamers aresingle-stranded DNA or RNA nucleotide sequences. Compared with antibodies,aptamers have the following advantages: high affinity and sensitivity with targets,stronger stability, short preparation period, little immunogenicity, wide range oftargets. Aptamers, as an alternative to antibodies, has shown bright prospects inscientific research, clinical diagnosis, and therapy.In the study, we first use dot blotting and Eastern blotting with aptamers todetect folded VEGF165protein and reducing VEGF165protein respectively, andcompare the results with antibodies. We also used a method called Aptamer-BasedRegionally Protected PCR (ARP-PCR) for VEGF165protein detection. The aptamerwas allowed to bind to the target protein in solution before digestion with DNase I.The region of the aptamer bound to the target was protected from DNase I cleavage.The target-binding region of the aptamer protected from the enzymatic treatment wasthen amplified by the PCR. At last we analysis the specificity of the ARP-PCRmethod.First we use Dot blotting and Eastern blotting to detect folded protein andreducing protein respectively, Positive signals were detected in the dot blotting for thetwo samples (1.75μg、3.5μgVEGF165), stating that sensitivity of dot blotting withaptamer and antibody is1.75μg and have a good specificity: while in the Easternblotting, the detect limit is14μg and also have a good specificity. But the sensitivityis less than dot blotting, maybe protein have some lose in the process of Electrophoresis and transfer membrane.We detect the protein with aptamer, in order to reflect the protection of proteinfor aptamer, the study was performed with and without protein. The result showedthat aptamer was all digested during0.5h-2h without protein, while aptamer wasprotected from digestion during0.5h-1.5h with protein and the more digestion time,the less brightness of amplification bands.The results of dot blotting and Eastern blotting with aptamer reflect that aptamerhas the equally sensitivity and specificity to antibody, so we could use aptamersubstituting antibody to detect proteins. In the method of ARP-PCR, protein couldprotect aptamer from DNase I cleavage, the digestion time is only0.5h, and have agood specificity. This method could detect protein quickly and conveniently, thedigestion time is only0.5h, improving the efficiency of detection.