| Cryptocaryon irritans is a parasitic ciliate that can parasitize on the skin and in the gills of almost all marine bony fish,which often causes the outbreak of cryptocaryoniosis in the epidemic area,and has brought serious harm to the mariculture industry.At present,there are no safe and effective methods for the prevention and cure.Studies have shown that cysteine proteases of the parasites play an important role in their whole life cycle.And can be used as one of the targets for preventing and treating parasitic diseases.In previous work,we have screened out a papain-like cysteine protease gene(CiPCP)from the c DNA library of C.irritans.And it was found that the recombinant protein expressed in E.coli had certain immunoprotective effect on Pseudosciaena crocea against the challenge of C.irritans infection.In this study,we detected whether the C.irritans gene could be expressed in P.crocea cells and yeast cells,and preliminarily evaluated the immunoprotective effect of the recombinant eukaryotic expression vector of CiPCP on P.crocea against the challenge of C.irritans.The results are as follows:1.we had isolated and cultured two cell lines of P.crocea gill(PCG)and brain(PCB).So far,the cells from the gill epithelial tissue and brain tissue of P.crocea have been passed on to 37 and 40 generations respectively,and they all grow well.The chromosome analysis showed that the chromosome number of the cultured cells was 48,consistent with that of P.crocea.These results indicate that the PCG and PCB cell lines of P.crocea have been successfully established.The growth of two kinds of cells in different medium was compared.It was found that the addition of 10% fetal bovine serum(FBS)to DME/F-12 was the best medium for PCG and PCB cells of P.crocea.2.We constructed the eukaryotic expression plasmids pcDNA/CiPCP and pcDNA/CiPCPt for the full-length and truncated papain-like protease genes(CiPCP and CiPCPt).The PCG and PCB cells of P.crocea were transfected with pcDNA/CiPCPt,pcDNA as negative control.The results of indirect fluorescent antibody assay showed that the gene of C.irritans could be expressed in the cells of P.crocea.3.The plasmids pcDNA/CiPCP,pcDNA/CiPCPt,pcDNA were injected into the muscle of the fish P.crocea respectively.After immunization,the fish was challenged with the infective theronts of C.irritans at the sublethal dose(3000 theronts/fish),and the immunoprotective rate in fish immunized with pcDNA/CiPCP or pcDNA/CiPCPt were31%and 26.6%,respectively.The results of real-time PCR showed that the expressions of interleukin-1βgene in liver,spleen and kidney of the fish immunized with pcDNA/CiPCP and pcDNA/CiPCPt were up-regulated,so were the expressions of IL-8A,Myd88 and TLR-8 in the fish liver.Meanwhile,the expression of Myd88 gene in kidney was also slightly up-regulated.This results showed that the pcDNA/CiPCP and pcDNA/CiPCPt had certain immunoprotective effects on the fish P.crocea against the challenge of C.irritans infection.4.In addition,the truncated gene(CiPCPt)of papain-like cysteine protease was inserted into Pichia pastoris expression vector pPICZ A,and successfully transferred electrically into yeast GS115 strain,the fusion protein r CiPCPt was then expressed after inducing with methanol.Generally,the recombinant protein expressed eukaryotically may have similar conformational structure with the native protein,which will faciliate the study for the active sites,properties and functions of the enzyme which is of great significance for further study on the control of C.irritans infection.In conclusion,two cell lines of P.crocea have been established in this project,which have not only proved that the foreign gene can be expressed in the cells of P.crocea,but also laid a foundation for further study on the interaction between host and pathogen,and in vitro culture of C.irritans.The results of the immunization trial have provided valuable references for the immunoprophylaxis of cryptocaryonosis in P.crocea. |
