Expression Of An Actin Gene From Cryptocaryon Irritans And Application Of The Actin And Its Related Molecules In Immunoprophylaxis

Cryptocaryon irritans is a species of parasitic ciliate causing cryptocaryonosis in marine teleost fish,which has brought about huge economic losses to the mariculture industry in China.It is urgent to explore the mechanism of the development and infection of C.irritans at the molecular level,and to use modern genetic engineering technology to find out the safe and effective prevention methods.Actin cytoskeleton are involved in cell division,movement,growth and shape maintenance.Actin depolymerization factor（ADF）binds to actin monomer（G-actin）by cutting off actin filament（F-Actin）,accelerating the conversion rate of actin in cells,which plays an important role in the dynamic changes of actin cytoskeleton.Profilin is also an important actin-binding protein,to a certain extent,which delays the aggregation of actin,making it delaying or aggregation more orderly.Profilin of some species can be used as adjuvant to enhance immune response.Studies have shown that cytoskeleton plays an important role in the process of parasite invasion and the transformation of different developmental stages,and plays a key role in directional cell movement.In order to explore the role of cytoskeleton-related protein of C.irritans in the process of invasion and transformation of different developmental stages,actin gene（CiActin）was screened out from the c DNA library of C.irritans trophozoite for recombinant expression and molecular characterization;then,a recombinant plasmid pcDNA/CiActin was constructed,and transfected into fishcellsto confirm the expression of C.irritans gene in fish cells.The recombinant protein or eukaryotic expression vectors of the above-mentioned molecules were used to immunize Pseudosciaena crocea for preliminary evaluation of their immunoprotection effects on P.crocea challenged with lethal or sublethal dosages of C.irritans.The experimental results are as follows:Prokaryotic expression and characterization of CiActin:the open reading frame ORF of CiActin（Gen Bank ID:JN399999）is 1131 bp and its theoretical molecular weight is 42 k Da.After the non-universal codons TAA and TAG encoding glutamine in the ciliate were transformed into the universal codons CAA and CAG,the whole ORF was synthesized.Then,CiActin was amplified and inserted into the vector,and the recombinant plasmid p GEX-4T-1/CiActin was constructed.The recombinant plasmid was transformed into E.coli DH5a strain,and the recombinant protein（rCiActin）was expressed after the addition of isopropy-β-D-thiogalactoside.The results of polyacrylamide gel electrophoresis showed that the molecular mass of rCiActin,expressed as inclusion bodies,was 68.0 k Da,which was consistent with the theoretical value.Then anti-rCiActin sera were prepared by immunizing mice（specific pathogens free）with rCiActin.Western blotting analysis with the sera showed that the endogenous CiActin was expressed in all development stages of C.irritans,and its molecular weight was consistent with the theoretical value.In addition,the results of indirect immunofluorescent antibody test showed that endogenous CiActin was mostly distributed around the plasma membrane and more concentrated in the cytostome of larvae.Expression of CiActin gene in the fish cells:fin tissues of S.marmoratus,which was used for the passage and conservation of C.irritans in our lab,were treated with trypsinase and cultured primarily.The epithelial cells and fibroblasts were successfully isolated and purified,and the chromosome numbers of the cells at the33^rdpassage were counted,both of which were 48,as same as that of the fish somatic cells Detection with indirect fluorescent antibody test showed that the fish cells transfeceted with pcDNA/CiActin expressed the foreign gene from C.irritans properly.The immunoprotective effect of the recombinant plasmid pcDNA/CiProfilin,pcDNA/CiActin,pcDNA/CiProfilin-CiActin on P.crocea suffered from the theront challenge were 49%,52%and 28%,respectively.Meanwhile that of pcDNA was 9%.The recombinant protein rCiADF₁was also used to immunize P.crocea.The immunoprotective effect of rCiADF₁on the fish suffered from challenge of C.irritans theronts was 59%,meanwhile that of the control GST was 9%.Comparison on the expression of the immune-related genes in fish liver,spleen and kidney among different groups of P.crocea:As detected by real-time PCR,the expressions of IL-1βin the livers and spleens of the fish in pcDNA/CiProfilin and pcDNA/CiProfilin-CiActin immunized groups was significantly up-regulated.The expressions of MHCII and IRF7 in the liver of the fish immunized with pcDNA/CiActin were slightly up-regulated;IL-1βwas up-regulated in the liver and spleen of the fish in rCiADF₁group.The results of immunization showed that rCiADF₁and CiActin,CiProfilin genes could induce the immune-related genes response of P.crocea.In conclusion,in this study molecular characterization of CiActin was carried out and the mouse antisera against rCiActin were prepared,which will faciliate the further study of actin cytoskeleton in the future.The fish cell lines established and transfeceted with pcDNA/CiActin expressed the foreign gene from C.irritans properly.The results of immunization showed that rCiADF₁and CiActin,CiProfilin genes could induce the immune response of P.crocea,and had certain immune protective effect on P.crocea,which provided a reference for the establishment of the strategies to control C.irritans infection.