Study On The Variation Of Complement Factor Ⅰ Treated With Maternal Immunity And Its Functionin In Ctenopharyngodon Idella

Grass carp(Ctenopharyngodon idella,C.idella)is a large variety of freshwater fish breeding species,but the healthy development of grass carp industry is long-term restricted by grass carp hemorrhage disease caused by grass carp reovirus(GCRV)infection.The major functional molecules of GCRV-resistance from C.idella show different viral resistance.To screen and confirm the major functional molecules of GCRV-resistance are play a significant role in molecular design breeding to produce C.idella of GCRV-resistance.In this study,to find the content variation of maternal immune factors in C.idella,investigate the regulatory role of CFI in GCRV-resistance immune response and the inhibitory effect in complement activation pathway,we explored the content variation of different immune factors(Ig M,C3,LZM,MBL,Bf,CFI)in C.idella treated with GCRV attenuated vaccine,and examined the structure characteristics,expression and biological function of CFI that was the only complement regulator with enzymatic activity in the complement activation pathway of C.idella.The results provide gene resources for molecular design breeding to produce C.idella of GCRV-resistance.The main results obtained are shown as follow:(1)The variation of six immune factors in C.idella treated with maternal immunity The proteins and m RNAs of six immune factors in C.idella immunized with GCRV attenuated vaccine were detected by ELISA and q RT-PCR,respectively.The results showed that the GCRV attenuated vaccine triggered a stronger response in C.idella and the expression levels of maternal immune factors increased significantly to defend against the invasion of external pathogens.Among them,the m RNA expression level of CFI was most significantly up-regulated,indicating that CFI may play an important role in the immune response defense against GCRV.(2)Structure and expression analysis of CFI in C.idella The bioinformatics analysis of CFI complete cDNA sequence obtained by NCBI in C.idella showed that CFI had a signal peptide containing 18 amino acids was defined at N-terminus.CFI protein included five domains of FIMAC,SR,LDLa1,LDLa2 and Tryp＿SPc and had a large number of cysteine conserved sites.The m RNAs and proteins of C.idella CFI detected by q RT-PCR and Western blot were expressed in tested tissues(liver,spleen,kidney,head kidney,intestine,gill,muscle and skin),with the highest expression level in liver.After GCRV infection,the m RNA and protein expression levels of CFI in the liver detected by q RT-PCR and Western blot showed a downward overall tendency,indicating that more CFI were used to lyse C3 b or C4 b,and inhibited the activation of complement system after GCRV stimulation in C.idella.(3)Functional study of CFI in C.idella By Constructing CFI(including signal peptide or not)vector and transfecting into GCO cells,the result showed that both the green fluorescence and the protein expression level of p EGFP-N1-CFI-NS(without signal peptide)were higher than p EGFP-N1-CFI-S(with signal peptide),confirming that C.idella CFI was a secreted protein and the removal of signal peptide was more beneficial to the accumulation of CFI in GCO.By constructing recombinant C.idella CFI protein and incubating it with C.idella serum,r CFI inhibited complement activation and degraded C3 b in serum,indicating that CFI in C.idella negatively regulates complement activation via degradation C3 b.To sum up,the expression levels of immune factors in C.idella can significantly increase treated with GCRV attenuated vaccine,but it has a certain time-effectiveness.Besides,these results provide the first evidence to indicate that CFI in C.idella negatively regulates complement activation via degradation C3 b,and probably plays a role in host immune defense against virus infection.