Heterologous Expression Of Daphnia Magna ChE And Applicability Of The Enzyme-based Antibodies

To prepare the genetic engineering antigen in a quick, convenient, and low cost way, a region (717bp) of genes of the ChE that belong to Daphnia magna were amplified with degenerate primers by RT-PCR. To construct recombinant vector, method of splicing was used for stringing genes of the ChE and expression vector PET-29a (+) by endonuclease digestion and ligation. The recombinant vector was then transformed into E.coli BL21(DE3) strain. The identified positive strain was proofed able to express ChE protein under the induction of IPTG The collected protein was denaturalized with urea, purified with immunoaffinity column, and then renaturalized by dialysis with renaruralizing solution. ChE activity of the protein was confirmed using PTch as substrate.Anti-serum of1:24000folds was obtained by immunizing BALB/c mice with purified protein of the recombinant ChE. To quantify immunoreaction content of ChE in Daphnia magna, indirect non-competitive enzyme-linked immunosorbent assay (ELISA) that used the mice anti-recombinant ChE as antibodies was established. The minimal detection limit of the method was estimated to be44.8ng·mL-1.The standard curve for testing of immunoreaction content of ChE (ChE-IR) was found in accord with the equation of Y=a·(1-e-bX) with a=1.082, b=0.291, r=0.9981, and S=0.025. The coefficient variation of the established ELISA was less than10%. Fortified recovery of the method was92.9-102%.Cross reaction of Daphnia magna and non-ChE Marker(5μg·mL-1,10μg·mL-1) with anti-serum was62.5%,0.08%, respectively; Specificity of anti-serum was found high, with cross reaction of33.33%,18.67%,5.33%,3.53%,3.07%,2.20%,2.33%,0.87%, and0.67%, towards, Milleri kiser,Chaenomeles sinensis,Bombyx mor, Chironomus kiinensis, Apis melliferaL, Brachydanio rerio (the brain and the whole body without brain), Eisenia foetida, and tadpoles ofXenopus laevis, respectively.To evaluate applicability of the genetic-engineering-enzyme-based non-competitive ELISA, the method was used to quantify the ChE-IR in Daphnia magna being exposed for21d to concentrations of triazophos ranging from1/7to1/329of immobilizing EC50or in those acclimatized to ambient temperature ranging from10.1℃t o21.3℃and in those to repeated freezing and thawing between-80℃and21.3℃. The tests indicated an36.40%-57.17%induction in terms of the content of the ChE-IR at2nd d, the variation tendency of ChE-IR was identical with the result previously(with polyclonal anti-serum of cholinesterase which was obtained by immunizing BALB/c mice with purified ChE from Daphnia magna)which proved the anti-serum were available; The rate of induction was25.63%and16.88%at10.1℃and15.9℃compared with that at25℃, respectively. This suggested that synthesis of the ChE sped up in anticholinesterase exposure and at low temperature. The result of multigelation test showed that in comparison with the control, the content of ChE-IR was not changed significantly, it is reasonable to infer that the content of ChE-IR was stable when Daphnia were freezed repeatedly between-80℃and2].3℃.