The Cloning Of Chitinase Gene In Daphnia And Preparation Of Polypeptide Antibody

Daphniidae plays an important role in maintaining the integrity and sustainability of food chains of freshwater ecosystems.In this paper,in order to prepare the chitinase antibody which can recognize Daphniidae,we successfully obtained the full-length chitinase sequences by RT-PCR and RACE method from Daphnia carinata and Simocephalus vetulus.The full-length of Daphnia carinata cDNA was 1404 bp and encoded 383 amino acids.While the full-length of Simocephalus vetulus cDNA was 1019 bp and encoded 382 amino acids.Based on their cDNA sequences,the genomic structures of the two chitinases were characterized.Sequence analysis revealed that DcChi was composed of three exons and two introns,while SvChi had four exons and three introns.Based on the results of multiple alignments of chitinases including these two chitinases,we designed four peptides that were specific to chitinases from the Family Daphniidae.They were ADHHAPLRKRS,GRQEPWQFGVENV,KRSFETTDNNVEYS,WEYPRSEADKTGF.The polyclonal antiserum of chitinase was successfully obtained by immunizing New Zealand white rabbits with synthetic artificial peptides as antigens.Then four antibody IgGs were obtained by purification and were named 001,002,003,004 antibody,respectively.In the study of the specificity of four kinds of antibodies,we extracted crude enzyme from six species of Daphnia carinata,Simocephalus vetulus,Dolerocypris sinensis,Eucyclops serrulatus,Caridina,Chironomus kiiensis and collected their free enzyme fluid in water respectively.The cross reaction results showed that 003 antibody could better recognize chitinase of Daphniidae both in vivo and in water,and had low cross-reactivity with other organisms(less than 6%)like Dolerocypris sinensis,Eucyclops serrulatus.Therefore,the 003 antibody prepared in this paper can be used to detect the population change of Daphniidae.