Screening,Characterization And Preliminary Application Of Nucleic Acid Aptamer For β-Conglycinin

β-conglycinin is one of the main allergenic proteins of soybean,which can cause severe symptoms of allergic reactions.Therefore,the efficient detection ofβ-conglycinin is particularly important.Although several allergen detection methods are available,such as mass spectrometry,immunological assays and polymerase chain reaction techniques,there are many problems with these methods that limit their widespread application in practice,such as high testing costs and complicated operations.Therefore,the establishment of a convenient,rapid and sensitive food allergen detection method can prevent sensitive people from food allergic reactions.Nucleic acid aptamers are single-stranded oligonucleotide sequences that bind to their targets with high affinity and specificity.The aptamers were obtained by systematic evolution of ligands by exponential enrichmen（SELEX）.Thus aptamers are called“chemical antibodies”and have a wide range of applications.The nucleic acid aptamer ofβ-conglycinin was screened using the subtractive-SELEX method in this study.Secondly,the sequence information was obtained by high-throughput sequencing technology,and the candidate aptamer sequence was characterized by bioinformatics technology and enzyme-linked aptamer method,and the optimal aptamer was obtained.Finally,we employed the aptamer as a recognition probe and nanogold as an indicator to develop a novel detection method forβ-conglycinin using an aptamer-nanogold colorimetric assay.The main research contents and results are as follows:（1）β-conglycinin nucleic acid aptamer was screened by subtractive-SELEX method.β-conglycinin,as a glycoprotein,can be immobilized to the enzyme plate by its hydrophobic property,setting up a screening method forβ-conglycinin targeted nucleic acid aptamer.In this paper,a library of oligonucleotides was synthesized in vitro,consisting of 20 bp primer sequences at both ends and 40 bp random base sequences in the middle.Next,the aptamer group that could bind specifically toβ-conglycinin was obtained by incubation steps.Next,the single-stranded DNA recovered by ethanol precipitation was used as a template and amplified by PCR to obtain the aptamer clusters obtained in each round of screening.After that,the magnetic bead method was employed to prepare the secondary library for the next screening round.The screening rounds were monitored by the recovery rate,and when the recovery rate reached 46.2%and didn’t continue to increase,the screening rounds were stopped as the number of rounds reached 10.The aptamer population was subjected to the high-throughput sequencing.（2）Based on the high-throughput sequencing results,the candidate obtained aptamer sequences were identified and analyzed.Firstly,high-throughput sequencing was performed on the screening products,and 39 sequences were obtained after selection,which contained several high-frequency sequences.Secondly,DNAMAN was used to analyze the homology.The results showed that there was no fixed sequence.Further,Mfold was utilized to conduct sequence-based simulations of secondary structure and predict their lowest free energy.The results showed that most of the secondary structures of the sequences were typical secondary structures such as stem and loop.Based on the principle of lower free energy,three nucleic acid aptamer sequences were selected for subsequent experiments.Finally,the affinity and specificity of the candidate nucleic acid aptamers were determined by the enzyme-linked aptamer method,and the findings indicated that the affinity and specificity of Seq-21 were better than the other two aptamers,so Seq-21 was selected as the optimal aptamer.（3）Development of a nanogold colorimetric method for visualization and detection ofβ-conglycinin based on optimal nucleic acid aptamer and nanogold agglomerative color change effect.The results of UV spectra showed that there is a maximum absorption peak at 520 nm,suggested that the nanogold solution was successfully synthesized.The detection parameters,i.e.sodium chloride and nucleic acid aptamer concentration,were optimized.The results showed that the color of the nanogold solution changed to gray when the sodium chloride concentration was 250 m M,and the absorbance value was stable when the aptamer concentration was 2 n M,which indicated that this condition was the optimal detection condition and could be used for the establishment of the subsequent detection method.Finally,based on the optimal aptamer concentration and nanogold agglomerative color change effect,a visualization method was established for the detection ofβ-conglycinin.Under optimized experimental conditions,the concentration ofβ-conglycinin exhibited a strong linear correlation with the A₆₂₀\A₅₂₀ values.The regression equation was y=0.37264+0.52545x,where R² was 0.98763,with a linear range of 0.08～1.70μg/m L and a detection limit of 0.0489μg/m L.In addition,the method showed a strong sepcificity forβ-conglycinin.The recoveries of the method in food matrices were 90.6～96.0%by spiked recovery test.Our results illustrate that the developed method is both straightforward and highly sensitive,exhibiting strong specificity.This method therefore provides a new point of reference for detectingβ-conglycinin in soybean.This method can serve as a valuable technical aid for allergy prevention and control,as well as the development of hypoallergenic foods.