Construction And Screeininc Of SCFV Phage Display Antibody Library Specific For Pyrethroid

The widespread use of pyrethroid insecticides led to the residue in water, tea and a lot of economic crops. And people have paid more attention to it. So the detection of pyrethroid insecticides has important significance in food safety. Currently the instrument operations have time-consuming and labor-intensive shortcomings, which make a great impact on their practicability, resulting in the occurrence of hysteresis in the market monitoring. The immunoassay method are widely used as a more advance detection of small molecules based on the advantages of the easy operation, simple pre-treatment process, low cost, rapid, accurate and sensitive detection, high throughput and automated testing, etc. In the immunoassay, the antibody is the key factor to determine the sensitivity and specificity. Genetic engineering antibody is a more advanced form of antibodies. The antibodies have a small molecular mass and the anomic acid sequences are known. It is easy to modify the genes of the antibodies and prepare a lot. And the cost is relatively lower than others. So genetic engineering antibodies become the focus of immunological research methods.In this study, total RNA was extracted from the Pyrethroid monoclonal cell saved in our lab, which have highly specific. And the cDNA was obtained by reverse transcription and used as the template for PCR amplification of variable regions of antibodies. The variable regions of light chain (VL) and heavy chain (VH) were about400bp in length respectively. Then the single-chain variable fragment (scFv) about780bp was obtained with overlap PCR method. The scFv fragments were digested by restriction enzyme EcoR I and Hind III after recycling and purification. The digestion production was ligated into T7phage arms and the Pyrethroid-specific phage-displaying antibody library was constructed. After examination original titer of the primary phage library reached2.67×105pfu. The selected phage clones were PCR amplified randomly. The results showed that the positive rate of antibody library is81%. The antibody library was panning using pyrethroid-OVA as the coating ligand. After4rounds of panning and enrichment, the specific phage clones was obtained.4positive clones were randomly selected and sequenced. Sequencing results indicated that the light chain variable fragment and the heavy chain variable fragment of pyrethroid single-chain variable fragment comprised108and119amino acids respectively. This study lays the foundation for preparation of a new form of antibody against Pyrethroid and innovation of immunological detection methods.