Construction Of Phage Display Antibody Library And Screening Of ScFv Against Carbaryl

Pesticide residues are harmful to human health; therefore detection of pesticide residues has become the research hotspot. ELISA is a well used method in pesticide residues detection. Antibodies play a key role in the method. Compared to classic polyclonal antibody and monoclonal antibody engineered antibodies have many advantages. In this study, T7phage was used to construct a naive murine antibody library and a carbaryl immune murine antibody library. Then specific single-chain antibodies (scFv) against carbaryl were screened from the library using ELISA method and recombinant expressed in E. coli. This work will give assistance to application of engineered antibodies in carbaryl detection with ELISA method.1mg total RNA were extract from the spleen of non-immunized Balb/C mice. Then the first chain of cDNA which derived from the purified mRNA by reverse transcription was used as template to get the VL and VH using degerate primers, and then connected to be the scFv with the SOE-PCR. With the EcoR I and Hind III double digestion the scFv was inserted into the T7phage vector arms and then been vitro packaged. At last we successfully constructed a naive murine phage library with the capacity of1.74×107pfu. We randomly selected10plaques for the PCR testing of the recombinant gene, and the correct recombinant rate reached90%. We used5plaques for gene analysis, and their sequences were different. The result proved the phage library had high quality and diversity. After the library liquid lysate amplification through infecting E. coli BLT5403, we used ELISA method to screening the anti-carbaryl scFv, but the antibody binding to carbaryl couldn’t been rich. The result showed the specificity of the naive phage with target was low, so that we constructed the carbaryl immune phage library for panning the anti-carbaryl antibody.Balb/C mice immunized with carbaryl antigen5H-KLH were used to construct immune T7phage display antibody library using same protocols to naive antibody library construction. The immune antibody library reached1.74×107pfu. Different amounts of carbaryl antigen5H-OVA (50,25,5,1μg/mL, respectively) were coated, and10pfu phages used for every round panning, and required1.08×105,2×104,3×106,3.2×106pfu/mL eluted phages respectively. We randomly selected58plaques from the4th round of eluted phages, and obtained19positive clones through PCR analysis. Direct competent ELISA was used to determine the specificity, and we put the scFv genes of two selected clones insert into plasmid pET26b, and then transformed into E. coli BL21(DE3) for expression with IPTG. The SDS-PAGE analysis results showed the clone A16-3expressed30kDa proteins successfully and we could obtain soluble scFv antibody with0.1and0.05mM IPTG in20℃. Purified scFv was tested the activity with the directed ELISA method. The IC%was18%when the coated antibody was1μg/well and the concentrate of carbaryl was16ppb. The result proved that the purified antibody had the binding activity and specificity to the carbaryl.