| Pyrethrins as one of six active esters (pyrethrinI/II, cinerinI/II and jasmolinI/II) from the flower extract of pyrethrum (Chrysanthemum Cineraraeflium) is the oldest known insecticide or pesticide for high efficiency, low toxin, broad-spectrum, and low residues. At present, the presence of pyrethrins can be determined by various methods, including gas chromatography (GC), high-performance liquid chromatography (HPLC) etc. Nevertheless, there are always setbacks and problems such as high request for equipment, harsh technically demanding and extensive sample processing prior to analysis, as well as incomptence for spot detection. Immunoassay is a prospective detection technique for the advantage of low-cost and fast detection on spot etc.Recombinant antibodies (rAbs) are a new immulogical test for many advantages, such as high affinity, high specificity, lower limit of quantitation and massive expression in prokaryotic system so on. Today, no information is currently available regarding anti-pyrethrins rAbs. Therefore, the development of the scFvs would be much valuable for the immunoassay or biosensor research of pyrethrins. In this study, we combine overlap PCR and subtractive phage display to construct recombinant scFv library from immunized mice, and then isolate monoclonal antibody with affinity for six esters of pyrethrins, which lays the foundation for further immunological analysis. The main results were as follows:①The scFv DNA library agaisnt pyrethrins was amplified by an optimal overlap PCR. First, the immunogen containing six antigens were used to immune BALB/c mice to prepare antiserum. The antibody titers were up to 105 by indirect ELISA. Second, the fisrt-strand cDNA was synthsized using purified mRNA from immunized spleen cells, and the VH and VL fragments were amplified by PCR indicating they were about 380 bp and 660 bp. Then two scFvs were spliced by overlap PCR, of which one with a long linker (Gly4Ser)3 was 780 bp, and the other with a short linker (Gly4Ser) was 750 bp.②The phage library specific for pyrethrins were constructed by phage display. First, the scFv and pComb3H were analyzed by sfi I, and the ligations were transformed into XL1-Blue for positive clones. Total size of the long-linker library was up to 1.3×107 and that of the short-linker library up to 2.6×107. Furthermore, phgage VCSM13 was used to prepare scFv phage library, which indicated the titer of the former was about 1×1012 cfu and the latter about 7.5×1012 cfu. ③The enrichment of scFv antibody library was performed by four rounds of successive screening. First of all, the long- and short-linker libraries were selected by four rounds of screening and the recoveries were up to 50 and 44 times, respectively. Moreover, ELISA was used to detect the effect of selection and the diversity of the library was analyzed by Bst NI enzyme, showing that the molecular diversity was about 40～50%.④Monoclonal scFv antibody against six esters of pyrethrins was isolated by ELISA. Total 96 clones from the last round of long-linker library were cultivated for screening individual clones against six esters of pyrethrins. The results indicated that 25 clones showed an anbody activity to pyrethrins, of which 3 and 5 clones were specific for pyrethrin I and II, 5 and 7clones specific for cinerin I and II, and 7 and 3 clones specific for jasmonlin I and II, respectively. Next, phage ELISA screening of 96 randomly selected output clones from the short-linker library produced 15 clones binding to pyrethin I and II. 9 clones showed the affinity for pyrethrin I and 7 clones exhibited the affinity for pyrethrin II. In addition, cross reactivity was observed in different esters of pyrethrins. |
