Identifications And Expression Analysis Of The Complement Alternative Pathway Related Genes In Megalobrama Amblycephala

Blunt snout bream(Megalobrama amblycephala)is one of the main freshwater fish cultured in China.However,bacterial septicemia caused by Aeromonas hydrophila is becoming more serious in recent years,resulting in huge economic losses.The acquired immune system of fish is underdeveloped,and innate immune system plays an irreplaceable role in resisting pathogen invasion.Complement system is an important part of innate immune system.Complement alternative pathway including complement factor D,factor P,factor Iand factor H,is the oldest complement activation pathway and plays an important role in the activation of complement system.The study of complement alternative pathway related molecules will help us understand the innate immune defense mechanism of the blunt snout bream,which is of great significance in disease control and genetic breeding of blunt snout bream.In this study,the ORF sequences of Pf,If and Hf genes in blunt snout bream(MamPf,MamIf and MamHf)were cloned and their sequences were analyzed by bioinformatics.Quantity real-time PCR(qPCR)was used to analyze the expression of MamPf,MamIf and MamHf genes in healthy tissues and after A.hydrophila infection.We obtained the MamDf recombinant protein by prokaryotic expression,and detected the bacteriostatic activity of MamDf recombinant protein.Western blot was carried out to detect the expression of MamDf protein in the liver,spleen,kidney and head kidney of blunt snout bream after infection with A.hydrophila.The main results of this study are as follows:1.Gene identifications and sequence analyses of 3 complement genes(MamPf,MamIf and MamHf)from blunt snout bream.The full-length opening reading frame(ORF)of the MamPf was 1329 bp,encoding 442 amino acids with a molecular mass of 48.9kDa.No N-glycosylation site was predicted.Multiple sequence alignment analysis showed that the MamPf and other species consisted of 6 TSR domains,and there were correspondingconserved sequences in the TSR domain.Phylogenetic analysis showed that fish Pf clustered into one branch and mammals clustered into another.The MamPfshowed the highest identity with zebrafish.The analysis of gene structure showed that MamPf gene contained 9 exons and 8 introns,and the number of exons and introns was similar among different species.The ORF of the MamIf was 2007 bp,encoding 668 amino acids with a molecular mass of 74.6kDa.6 N-glycosylation sites were predicted.Multiple sequence alignment analysis showed that the MamIf and other species consisted of one FIMAC domain,one SRCR domain,two LDLa domains and one serine protease trypsin superfamily domain,and conserved sequences were found in each domain of different species.Phylogenetic analysis showed that fish If clustered into one branch and mammals clustered into another.The MamIfshowed the highest identity with carp.The ORF of the MamHf was 2589 bp,encoding 862 amino acids with a molecular mass of 97.5kDa.2 N-glycosylation sites were predicted.MamHf gene contained 13 repetitive SCR domains,and the number of SCR domains in different species was different.Phylogenetic analysis showed that fish Hf clustered into one branch and mammals clustered into another.The MamHfshowed the highest identity with zebrafish.2.Expression of three blunt snout bream complement genes(MamPf,MamIf and MamHf)in healthy tissues and main immune tissues after infection with A.hydrophilaThe quantity real-time PCRresults showed that MamPf,MamIf and MamHfgenes were expressed in 10 tissues.The MamPf gene was highest expressed in the kidney,lowest expressed in the intestine and also dectected in other tissues.The MamIf gene was highest expressed in the liver,high in the kidney,blood,gill and heart,and lowest expressed in the brain.The MamHf gene was highest expressed in the liver,lowest expressed in the muscle and also dectected in other tissues.After infection with A.hydrophila,the MamPf,MamIf and MamHfmRNAs changed significantly.In the liver,MamPf was significantly up-regulated and peaked at 24 h after infection,and returned to the control level at 5 d.MamIf was significantly up-regulated at 4 h,and peaked at 5 d.MamHf was significantly up-regulated from 4 h to 5 d,and peaked at 3 d.In the spleen,MamPfwas significantly down-regulated at 4 h,and significantly up-regulated and peaked at 24 h.MamIf was significantly up-regulated and peaked at 4 h,then decreased to the control level.MamHf was significantly up-regulated and peaked at 4h,and decreased to the control level at 24 h.In the kidney,MamPf was significantly up-regulated from 4 h to 5 d,and peaked at 4 h.MamIf was significantly up-regulated at 4 h and peaked at 24 h,subsequentlysignificantly down-regulated at 24 h,and decreased to the control group at 3 d.MamHf was significantly upregulated at 4h and 24 h and peaked at 4h.In the head-kidney,MamPfwas significantly up-regulated from 4 h to 3 d,and rpeaked at 4 h,and decreased to the control level at 5 d.MamIfwas significantly up-regulated and peakedat 4h,subsequently significantly down-regulated,and then reached the control level at 5 d.MamHf was significantly up-regulated at 4 h and 24 h and peaked at 4 h,and returned to the control group after 3 d.3.Antimicrobial activity analysis of MamDfrecombinant protein and Western blot AnalysisThe MamDf gene had 5 exons and 4 introns.The prokaryotic expression vector pET28a-Df was successfully constructed,and the optimal expression conditions(37 ℃,180 r/min,IPTG=0.05 mmol/L induction for 6 h)were screened.The recombinant protein of MamDf had obvious inhibitory effects on the growth of A.hydrophila,Escherichia coli and Staphylococcus aureus,and the inhibitory effect on gram-negative bacteria was greater than that against gram-positive bacteria.Western blot analysis in immune tissues showed that MamDf protein was up-regulated in liver,spleen,kidney and head-kidney within 4 h-5 d after A.hydrophila infection.