Studies On New Fluorescence Method Based On RecA Fusion Protein Inducing Mechanism

Fusion protein is an exogenous protein which was expressed by prokaryotic cells,eukaryotic cells,insect cells and animal cells.Although the fusion protein is not the original protein of the cell,the cell expression process is not recognized,resulting in the genes in the expression fragment can be fully expressed.In order to facilitate research in the field of medicine and chemistry,specific bioactive proteins can be artificially designed.The fusion protein could be used for the study of structure and function.Therefore,with the advantage of fusion protein,this paper designed the Rec A-GFP fusion protein to develop some novel fluorescent strategy and apply Rec A-GFP fusion protein in DNA and telomerase fluorescence detection.Deoxyribonucleic acid(DNA)is indispensable to life,referred to as the genetic material in the body.It controls the synthesis of protein and the function of the organism.Individual body function is closely connected with the expression of DNA.The change of DNA structure will have a significant impact on living organisms,which is associated with the cause of a variety of diseases.Mutations DNA Detection is of great significance for early diagnosis and treatment of genetic diseases.Similarly,telomerase is considered a cancer biomarker for early cancer diagnosis.Telomerase elongates the telomeres of cancer cells,which leads them to unlimited replication potential.Therefore,the development of telomerase detection method is of great significance for cancer prevention and treatment.The paper is divided into three parts:1.A Simple,Specifically Fluorescence Method for Detection of DNA by Using RecA-GFP Fusion ProteinThe bio-modified single-strand DNA and Rec A-GFP fusion proteins were conjugated with streptavidin magnetic beads to form the magnetic fluorescent probes.In the present of double-stranded DNA which was containing homologous sequence,single-stranded DNA searched for homology from double-stranded DNA,and then catalyzed the exchange of the complementary strand,producing a new heteroduplex by the help of RecA-GFP fusion protein.During homologous recombination,strand invasion at one end and RecA-GFP fusion protein dissociation at the other end occurred.Because of the function of RecA-GFP fusion protein,we establish a fluorescence analysis method for rapid detection of double-stranded DNA.The reaction conditions were optimized.Under the optimal experimental conditions,the linear range for detection of DNA was from 1nM to 11 nM.The linear equation was ΔF=169.28C+251.55.The detection limit is 0.3 nM.2.“Turn-On” for the Highly Selective Detection of DNA in Living Cells Based on fluorescence resonance energy transfer ProbeHerein,this study designs a probe termed fluorescence resonance energy transfer probe to achieve fluorescent detection of intracellular DNA with higher specificity.The probe is composed of a gold nanoparticle,single-stranded oligonucleotides and Rec A-GFP fusion protein.In the presence of homologous DNA,DN A strand invasion at one end and Rec A-GFP fusion protein dissociation at the other end occurred,so that Rec A-GFP fusion protein is free from gold nanoparticle,resulting in high fluorescence.The reaction conditions were optimized.Under the optimal experimental conditions,there was a good linear relationship between the fluorescence restoration and the number of E.coli cells.The linear range for detection was from 0.05 OD to 1 OD.The linear equation was ΔF=169.28C+251.55.The detection limit is 0.3 nM.3.A Simple,Sensitive,Label-free Fluorescence Method for Detection of Telomerase Activity Using RecA-GFP Fusion ProteinThe telomerase substrate primer was conjugated on the surface of microplate through interaction of streptavidin-biotin.In the presence of telomerase and dNTPs,the primer could be extended.TTAGGG repeat units were continuously added to the 3’-end to form a long telomeric DNA.Then,numerous Rec A-GFP fusion proteins wrapped around the telomerase elongation products,which resulting in high fluorescence intensity.We develop a simple,ultrahigh sensitivity and label-free method for detection of telomerase activity,which relied on that fusion proteins wrapped around telomeric DNA.The reaction conditions were optimized.Under the optimal experimental conditions,there was a good linear relationship between the fluorescence restoration and the number of He La cells.The linear range for detection was from 50 to 1000 cells.The linear equation was ΔF=1.28C+142.4.The detection limit is 8 HeLa cells.