The Research Of Brcaa1 Gene And The Effects Of The Nanoparticles On Reaction System

Aim: To prepare the BRCAA1 short peptide including novel antigen epitope with bioactivity. To explore the effects of mercaptoacetic acid (TGA) modified CdTe quantum dots on the specificity of PCR,and the effects of different sizes of Au nanoparticles on efficiency of rapid translation system (RTS).Methods: The primers with his tag for amplifying brcaa1 gene fragment including novel antigen epitope were designed according to the brcaa1 sequence(AF208045), the gene fragments were obtained by Polymerase Chain Reaction based on use of brcaa1 plasmid as template, and confirmed by sequencing. The short peptides including novel antigen epitope were obtained by Rapid Translation System(RTS), and its antigen activity was identified by Western Blotting. TGA modified CdTe quantum dots were added into PCR liquids, brcaa1 gene vector was used as PCR template, two-round PCRs were done. The final PCR products were analyzed by 1% agarose gel electrophoresis, Photoluminescence (PL) spectroscopy, X-ray photoelectronic spectroscopy (XPS), and UV-vis spectroscopy. Au nanoparticles of different sizes were added into RTS system, using brcaa1 gene fragment as template, reacting at 30°C for 6h, then the protein products were quantified with ND1000 instrument.Results: The gene fragments including novel antigen epitope were obtained and confirmed by PCR and sequencing analysis. The matched short peptides were fabricated by RTS. Western blotting confirmed that the matched short peptides owned antigen activity. In the range of less than 1.33 mg/ml TGA-modified CdTe quantum dots, as the amounts of quantum dots increased in the PCR liquids, the non-specific amplification bands gradually disappeared. XPS showed that no element position shift was observed, UV-vis spectroscopy analysis showed that UV-vis absorption spectra after PCR was higher than that before PCR. In the RTS, adding gold nanopartilces with 5nm with the scope of less than 1.2nM, the efficiency of RTS can be markedly enhanced. Conversely, the Au nanoparticles of 10nm and 20 nm in size added into RTS reaction liquids, the efficiency of RTS can be decreased.Conclusion: The short peptides including novel antigen epitope with antigen activity are fabricated successfully, which lays foundation for further antibody preparation and diagnosis method establishment. Addition of TGA modified CdTe quantum dots in the range of less than 1.33mg/ml into PCR liquids can markedly improve PCR specificity. The phenomena owns potential in application such as ultra-sensitive DNA detection, photoelectric biosensors. Addition of 1.2nM Au nanoparticles of 5nm in size into RTS can markedly improve the efficiency of RTS.