The HcCnAα gene cloning in Hyriopsis cumingii and expression after infection with Aeromonas veronii
Calcineurin(CN)is a threonine/serine protein phosphatase that plays an important role in the antimicro-bial response and immune response in molluscs.In order to investigate the role of CN in the antimicrobial immune response of Hyriopsis cumingii,the α-type catalytic subunit of H.cumingii CN gene(HcCnAα)was cloned by rapid-amplification of cDNA ends(RACE)and its characteristics were analyzed by bioinformatics methods.With the pET30a-HcCnAα expression vector successfully constructed and the mice immunized after protein purification and ultrafiltration,polyclonal antibody with good specificity was obtained.The relative expression of HcCnAα in different tissues were analyzed by Western blot(WB)and quantitative real-time PCR(qPCR)in a fit state or infec-ted by Aeromonas veronii GL2.The results showed that the full length of HcCnAα cDNA was 2 737 bp,includ-ing 131 bp of 3'UTR,1 154 bp of 5'UTR,and it encoded a total of 483 amino acids.The sequence contained PP2Ac domains,which are conserved domains of protein phosphatase.WB and qPCR results showed HcCnAαwas expressed in all the tissues of H.cumingii.The expression of HcCnAα was highest in adductor,higher in foot,gill,intestines,kidney,and gonad,and the lowest in blood.After infection with A.veronii GL2,the expression of HcCnAα was significantly up-regulated in gills at 3 to 24 h,and also in adductor at 6 h,in hepatopancreas,foot,blood,and mantle at 24 h,but the expression significantly decreased in adductor,gill,mantle,and hepatopancreas at 48 h.This study suggested that the HcCnAα gene of H.cumingii was highly similar to the CnAα of other species with conserved PP2Ac domains;it was expressed in all tissues,and played a role in immune responses after the bacterial infection.This study successfully accomplished the cloning of HcCnAα genes for the first time and the polyclonal antibody was obtained,supporting the involvement of HcCnAα in the immune response after the bac-terial infection in H.cumingii,and thus established a solid groundwork for future investigations in immune regula-tion,disease prevention and control strategies for H.cumingii.
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维普
