Preparation And Application Of Antibodies For Recombinant Chitinase From Daphnia Carinata And Simocephalus Vetulus

For preparing the antibodies efficiently,the genes of the chitinase（EC 3.2.1.14）that belong to Daphnia carinata（1149bp）and Simocephalus vetulus（1086bp）were amplified by RT-PCR.To construct recombinant vectors,the chitinase genes and the expression vector pCold were both digested and ligated by endonuclease.The recombinant vectors were then transformed into E.coli BL21 strain and the later were induced by IPTG,and the chitinase protein of two the species was successfully expressed.Antibodies of 1:158000 and 1:134000 folds were obtain by immunizing New Zealand White Rabbits with purified the recombinant protein of chitinase of Daphnia carinata and Simocephalus vetulus.Indirect non-competitive enzyme-linked immunosorbent assay（ELISA）was used to determine optinum concentrations of antibodies and antigenes.For Daphnia carinata,the optimum antibody concentration was 4.75×10^-5mg·mL^-1,with the standard curve for testing of recombinant chitinase was y=1.4711×(1-e^-1.5431x)(R=0.9892,S=0.0915,minimal detection limit=24.1ng·mL^-1).For Simocephalus vetulus,the optimum antibody concentration was 4.36×10^-5mg·mL^-1,with the standard curve for testing of recombinant chitinase was y=1.8204×(1-e^-0.8393x)(R=0.9854,S=0.125,minimal detection limit=35.2ng·mL^-1).Reference to the antigenic species,cross-reactions of the two antibodies towards several species of crustacean（Daphnia carinata,Simocephalus vetulus,Neocaridina palmata,Eucylops serrulatu and Dolerocypris sinensis）with were tested.For Daphnia carinata’s chitinase antibody,except for Simocephalus vetulus,which was 10.46%,the cross-reaction rate with other species of crustacean were less than 0.28%.For Simocephalus vetulus’s chitinase antibody,except for Daphnia carinata,which was20.26%,the cross-reaction rate with other species of crustacean were less than 0.24%.A mixed population of Daphnia carinata and Dolerocypris sinensis was established and tested by chlorpyrifos.Population density（abundance）,specific activity of free-living N-Acetyl-β-D-glucosaminidase（NAGase）,and immunoreactive content of free-living chitinase（chitinase-IR）were measured.The chitinase-IR was quantified by gene-engineering antibodies developed by the prokaryotic expressed chitinase of the Daphnia carinata.No observed effect concentration（NOEC）and lowest observed effect concentration（LOEC）for population density,as they were scaled by actual concentrations,were 0.075 and 0.172μg·L^-11 for the Daphnia carinata and 0.014 and0.051μg·L^-11 for the Simocephalus vetulus.Community level（CaseR.1）NOEC and LOEC were less than 0.084 and less than 0.200μg·L^-1,respectively.The NOEC and LOEC were 0.061 and 0.133μg·L^-11 for the chitinase-IR and they were less than 0.041and less than 0.084μg·L^-1 for the specific activity of NAGase.The results demonstrated that chitinase-IR could be employed for indicating change of the population caused by an insecticide,and it provides a method reference for pesticide risk assessment of aquatic ecosystem.