The Immune Effect Of The Surface Antigen CiSA32.6t Of Cryptocaryon Irritans On Larimichthys Crocea Against The Parasite Challenge

Larimichthys crocea is one of the four major marine products in China and plays an important role in fishery economy.In recent years,with the continuous development of aquaculture and the increase of fish culture density,which facilitate the transmission of Cryptocaryon irritans,marine ‘white spot disease’ outbreaks frequently and at large-scale,which has caused great economic losses in the cultivation of L.crocea.And there is no safe and effective method to control the disease.Previous studies have shown that C.irritans can induce protective immune responses in the host.Therefore,whether genetic engineering vaccines,especially DNA vaccines and subunit vaccines,have good prospects in the control of cryptocaryonosis or not,has attracted our attention.Surface antigens of parasitic protozoa are considered to be the main immunodominant antigens,which participate in the interaction between parasites and host,and may be one of the main targets of immune prevention.Therefore,we preliminarily evaluated the immunoprotective effect of a recombinant truncated surface protein expressed prokaryotically and a recombinant plasmid for eukaryotic expression of the DNA fragment encoding the surface protein on L.crocea against the challenge of the sublethal dose of C.irritans infective larvae.For evaluating the antibody dynamics before and after immunization,mouse antisera against a recombinant truncated Ig M protein was also prepared,with which an enzyme-linked immunosorbant assay was established.The results are as follows:Construction of the prokaryotic expression system of immunoglobulin Ig M of L.crocea: In order to detect the dynamics of antibody levels before and after immunization,a Ig M gene sequence of L.crocea(Gen Bank: FJ589726.1)was obtained from NCBI database.And the prokaryotic expression system for the constant region fragment of the immunoglobulin Ig M heavy chain(Lc Ig Ht)of L.crocea was constructed.Anti-sera were prepared by immunizing mouse with the recombinant protein r Lc Ig Ht.Western blotting analysis showed that r Lc Ig Ht had good immunogenicity and reactivity.The sera of r Lc Ig Ht-immunized mouse could specifically recognize the native Ig M protein in the serum of L.crocea.Meanwhile,the recombinant protein r Lc Ig Ht can also be recognized by the sera from mouse immunized with native Ig M.The highest titer of the antibody in the sera of mice immunized with r Lc Ig Ht reached 102400,which laid a foundation for the immunodiagnosis of C.irritans infection and the evaluation of immunoprotective effect.The prokaryotic expression of the truncated C.irritans surface antigen(Ci SA32.6t):a prokaryotic expression system of Ci SA32.6t was constructed using p ET28 a as a vector.Ci SA32.6t is composed of 852 bases,encoding 283 amino acids with a theoretical molecular weight of 29.9 k Da.The recombinant protein r Ci SA32.6t has polyhistidine tag which is about 6 k Da.Compared with the GST fusion protein expressed earlier in our laboratory,the recombinant protein r Ci SA32.6t may be less affected by the tag protein and can expose more antigenic epitopes.Western Blotting analysis showed that sera from mice immunized with the soluble extract of C.irritans trophonts lysate bound to the recombinant protein.In addition,the eukaryotic expression plasmid pc DNA/Ci SA32.6t was constructed.The plasmid was transfected into the cultured cells preliminarily isolated from the gill and brain of L.crocea.The results of immunofluorescent antibody assay showed that Ci SA32.6t was successfully expressed in the cells of L.crocea.The preparation of r Ci SA32.6t and pc DNA/Ci SA32.6t provided materials for the further study.The recombinant protein r Ci SA32.6t and plasmid pc DNA/Ci SA32.6t were used as vaccine candidates to immunize L.crocea.Their relative percent survival against sublethal dose-C.irritans infection were 50.10% and 36.26% respectively.which preliminarily proved that these two vaccine candidates had certain immune protection function for L.crocea against the challenge of sublethal dose of C.irritans infection.The expressions of IL-1beta and Ig M were up-regulated in varying degrees,while MHC II showed no significant changes after injection of two candidate vaccines by RT-PCR.In conclusion,this study provides basic information for the serological diagnosis of C.irritans infection in L.crocea and the further development of subsequent immune control methods.