Construction And Application Of Two-dimensional Nanobiosensor Based On Bacterial S Layer

Biosensor is a sensitive inductive analysis device,which is widely used in clinical diagnosis,food safety,environmental monitoring and other fields.The bio-sensitive element determines the sensitivity of the biosensor.The fixation methods commonly used of bio-sensitive elements will reduce the activity of biomolecules,and the biomolecules cannot be arranged in order,which will also reduce the sensitivity.How to effectively fix bio-sensitive element on solid substrates and improve their sensitivity has always been the focus of biosensor research.The S layer is a two-dimensional protein array that is ubiquitous on the surface of many bacteria and archaea.It can self-assemble into the same lattice structure as the natural S layer on various interfaces in vitro,and is widely used to be the bridge of sensor carrier and the biological sensitive element,Through fusimg with the S-layer protein,the target protein can self-assembles on the sensor carrier surface and forms an ordered arrangement structure.However,S-layer proteins may lose self-assembly properties after fusion with certain proteins,so this construction method is not universal.This study proposes a strategy of fusion expression of S-layer protein with Spy Catcher,fusing target protein with Spy Tag.S-layer protein expressed by fusion with Spy Catcher has good self-assembly ability in vitro,while the target protein fused with Spy Tag can be covalently connected to the S layer protein,forming the sensitive element of a two-dimensional biosensor.The sensor abandons the fixation method of gold substrate and biomolecule through chemical cross-linking.At the same time,under the strong covalent binding of Spy Catcher-Spy Tag,the target protein can be fixed orderly with high density.Because the size of Spy Tag is only 13amino acids,it basically does not affect the structure and function of the target protein,and it is easier to perform cloning operations.Therefore,the construction method of the two-dimensional nanobiosensor proposed in this study is suitable for most target proteins to fixed softly on the surface of gold chips,keeping the activity of the target protein to the maximum,but also realizes the regular arrangement of the target protein on the sensor carrier.Using this two-dimensional nanobiosensor,combined with a surface plasmon resonance sensor,this study achieved high sensitivity detection of maltose binding protein(MBP)antibodies and preliminary screening of antibodies against African swine fever virus in swine serum The mian results are as follows1.The fusing protein r Sbp A-Spy Catcher was cloned and expressed in Escherichia coli and the lattice structure formed by Sbp A-Spy Catcher was observed under transmission electron microscope.2.The electrical impedance method was used to characterize the assembly of the fusion protein r Sbp A-Spy Catcher on the surface of the gold electrode.The results showed that the protein could be assembled well on the gold electrode surface.3.The maltose binding protein(MBP)is fused with Spy Tag,then expressed and purified in E.coli.Fix the r Sbp A-Spy Catcher on the surface of the gold chip,and then fix the Spy Tag-MBP to complete the construction of the two-dimensional nanobiosensor.Then perfore highly sensitive detection of maltose binding protein(MBP)antibodies.The results show that the lowest MBP antibody concentration the sensor can detect is 156 ng/m L,K_D=1.26×10^-13.4.The African swine fever virus proteins K78R,I215L,E146L were fused with Spy Tag respectively,then expressed and purified in E.coli.Follow the same method as above to construct a two-dimensional nano-biosensor against African swine fever virus protein antibody,and then use the swine serum infected with African swine fever virus as a test sample to detect whether there is interaction with K78R,E146L,I215L in the serum To evaluate their immunogenicity.The results showed that K78R was the most immunogenic,followed by I215L,and E146L was the weakest.This study not only provides a general strategy for the efficient,orderly,and gentle immobilization of biomolecules on biosensing surfaces,but also provides new ideas for the research of immunogenicity of important pathogens,which can assist in the development of related vaccines,screening of neutralizing antibodies,etc.