Heterologous Expression Of Chitinase In Dolerocypris Sinensis And Its Applicability For Antibodies Preparation

As a biological indicator,chitinase is often used in the aquatic ecological risk assessment of pesticides,especially the effects of pesticides on the development of crustacean.The ostracod is an important part of zooplankton,especially the most common species of benthic arthropods.The abundance of the worms,to a certain extent,can often reveal the health of a certain water area.Studies have shown that the content of chitinase in water has a significant correlation with the abundance of planktonic arthropods.In order to efficiently,conveniently and specifically measure the chitinase content of the water-mediated worm,we need to develop an antibody that specifically recognizes the chitinase chitinase.In this paper,the cDNA and gDNA full-length sequences of chitinase gene were cloned from Dolerocypris sinensis by RT-PCR and RACE.The results showed that the D.sinensis chitinase gene(DsChi)consisted of 10 exons and 9 introns,and the open reading frame was 1821 bp,encoding 606 amino acids.The recombinant plasmid was successfully constructed by ligating the gene sequence and the expression vector(pET-30a(+)).After the recombinant expression vector was transformed into E.coli,the positive strain was induced by IPTG to express the recombinant chitinase protein.The purified recombinant chitinase protein was used as antigen to immunize New Zealand white rabbits.The rabbit anti-D.sinensis chitinase antiserum was successfully prepared and purified to obtain the corresponding antibody.In the specificity study of antibodies,we extracted crude enzymes from ten species of Neocaridina denticulate?Neocaridina palmata?Daphnia carinata?Simocephalus vetulus?Aquarium paludum Fabricius?Ischnura asiatica?Chironomid plumosus?Cipangopaludina chinensis Gray?Carassius auratus auratus.The liquid was cross-reacted,and the results showed that the antibody could recognize the chitinase in D.sinensis,and the cross-reaction with other organisms was below 20%.Therefore,the rabbit anti-D.sinensis polyclonal antibody prepared by the present method can be applied to detect the change of the ostracod population in water.The research results provide a new method for identifying aquatic hazards of pesticides by using chitinase.