Construction Of ScFv Library And Screening Of Anti-nonylphenol Antibody By Ribosome Display

As one of the Environmental Endocrine Disrupting Chemicals(EDCs), monitoring and evaluation of Nonylphenol is importent in area survey.Although it is accurate by using instrument,these methods are time-consuming, laborious and costly. The rapid biological detection technologies such as immunological detection is based on the specific antibody of the small molecule pollutants. Recombination monoclonal antibody(RMA), since the 1990s, is a kind of antibody that antibody gene is reformed by cutting, splicing,modified, or synthesisised directly, then transformed into living cells for expression. Its chemical constitution is same as the monoclonal antibody, and remins stable immune specificity. Among all of these engineering antibodies, single chain variable fragment (ScFv) has been used in medical domain with characters of low or no immunogenicity, small molecular weight, powerful tissue penetration, low cost, full scale operation and so on. ScFvs can be obtained by phage display, ribosome display etc.. Because of entirely in vitro, ribosome display is far greater than phage display library in library capicity, and is simple in construction and screening with no selection pressure, which would enhance the affinity of targeting protein by the introduction of mutation and recombination. These advantages have made ribosome display technology demonstrate the attractive prospects.Utilizing the large capacity (up to 1013-1015) and the versatility of native antibody library of ribosome display, we extracted the total RNA from the mouse in different stains(Balb/C, C57) and synthetized the first chain cDNA by reverse transcription. After designing more appropriate primers to amplify the VH and VL gene fragments(about 350 bp) ,ribosomal display elements T (93 bp) was synthesized in vitro and interval sequence P (294 bp) from the vector PT7PD, we spliced these four fragments by overlap extention PCR. Firstly, ribosome display components T and heavy chain H were spliced;secondly, interval sequence P and light chain L were spliced, at last, the fragment TH and LP were spliced by the linker (Gly3Ser) 4 after the purification respectively and Ribosome display antibody library have been constructed which was about 1100bps then we identificated the activity of transcription, reverse transcription and translation of this library in vitro.After transcription and translation of the ScFvs library in vitro, we got the antibody-ribosome-mRNA(ARM)complex. Targeted by nonylphenol(NP-BSA), this complex was screened by solid affinity selection. We degraded the concentration of Mg2+ to elute mRNA which was used for in reverse transcription to create DNA library for next round. After several rounds of selection. specific antibody was finally obtained.The antibody was expressed in Escherichia coli and then purified. Because the versatility of native antibody library, theoretically specific antibody can be selected for any kinds of antigen using as targets, This methods established the foundation that we can select specific antibodies of any antigens especially micromolecule haptens.