The Initial Research On The Ribosome Display Technology Of Selecting The SEB Antibody

ObjectiveThe ribosome display library is established for selecting SEB antibody.MethodThe first part of the experiment is about construction of prokaryotic expression plasmid which can expresse the Staphylococcus aureus enterotoxin B protein. PET 32a is the vector, which is one of the advanced prokaryotic expression system of Invitrogen company . SEB gene and PET 32a vector are connected, then there is a expression of the SEB protein with 6 His tag in the N terminal under a suitable condition. HiTrap^TM Chelating is one of a range of pre-packed, ready-to-use, columns for preparative metal chelate affinity chromatograpy. We use a Ni-affinity column to purify the SEB which contains 6 histidine residues. Also ,we use Western blot in identification to determine the antigenicity of SEB protein.In the second part of the experiment, the RNA from the spleen of the C57mice and Balb/c mice is extracted firstly. The scfv region is constructed from heavy chain variable region gene (VH ) and light chain variable region gene(VL). Mice heavy chain variable region and light chain variable region is amplificated employing PCR technology , and then all the required components of ribosome display are connected in order to build a gene template. This gene template contains T7 promoter,5' loop,ribosome binding site,SCFV gene library,space,3'loop. After that the work of in vitro transcription and translation are carried out. Finally the ribosome display library is established for selecting and screening SEB antibody.ResultThe experiment has constructed the prokaryotic expression plasmid which can expresse the SEB protein successfully, and it is named SEB321. With 3mmol/L IPTG, we raises the SEB protein in 30℃for 4 hours. After getting the SEB protein, the experiment use a Ni-affinity column to purify the SEB protein and use Western blot to determine its antigenicity. The experiment has constructed the ribosome display library from mice scfv region. The determination of the library volume is 7.33×10¹³. Subsequently, the library in vitro transcription and translation proves that it can effectively carry out transcription in vitro. And the result of screening in ribosome display library with Staphylococcus aureus enterotoxin B as an antigen is positive.ConclutionIn this experiment, we have estabulished the ribosome display library from mice scfv region, and the library volume is 7.33×10¹³. The experiment uses this ribosome display library in the selecting of SEB antibody, the result is positive. The experiment get the SEB protein successsfully using the pET32a as a prokaryotic expression vector.