Identification 、 Recombinant Expression And Functional Analysis Of Complement Factor I And C3-1 Gene In Half-smooth Tongue Sole(cynoglossus Semilaevis)

Complement（complement, C） system was an important component for antimicrobial infection, which appeared early in the evolution of organism immune system. It was the most complicated restrictive protein dissolution system and consisted of more than 35 species of pyrolysis of enzymes, inhibitory enzyme factors and receptors. Complement system could be activated though classical pathway（CCP）, alternative pathway（ACP） and lectin pathway（LCP）, it could recongnize the foreign pathogen invasion signal, and participate in specific and non- specific immune response. Therefore, complement system play a significant role in congenital and acquired immunity. Complement factor I（Cfi） was a significant component of complement system, and main synthesized in the liver and play the role of the immune regulation. It could adjust the excessive activation of the complement system though enzymolysis C3 b and C4 b with the cofactors C4 bp, MCP and Cfh.Complement 3（C3） was a crucial component of complement system;it was the highest element in serum andplays thevital role in congenital immune and adaptive immune system.As the common molecule in threeactive pathways, C3 was mainly synthesized inmacrophage and liver. It could crack into C3 a and C3 b with the participant of C3 convertase. And then, under the stimulus of antigen-antibody complex or pathogene, C3 could shape the membrane attack complex（MAC） in the surface of the target cells, and crack the target cells finally.Cynoglossus semilaevis was an important variety and extensive breeded inoffshore fishing grounds with its huge economic value. However, healthy aquaculture industry of cynoglossus semilaevis breed has been limited by disease problem to some certain extend, especially the bacterial disease. In recent years, our labtory developed the research of cynoglossus semilaevis complement system. In this IX study, we identified, prokaryotic expressedand analyzed the function of recombinant protein, and cloned,sequenced and investigated the transcripts of complement component 3-1（C3-1） post infected of vibrio anguillarum. 1. Analysis of Cfi gene 1.1. Indetified andsequence analysis of Cfi geneOn the base of the builted genome-wide c DNA library andthe previous research of our laboratory from half smooth tongue sole.In this study, though cloned and sequenced, the full-length c DNA of Cs Cfi was accepted, it was 2230 bp in length, including a 98 bp 5’- untranslated region（UTR）, a 164 bp 3’-UTR and a 1968 bp open reading frame（ORF）. It encoded a polypeptide of 656 amino acids, with a molecular mass of 72.28 k Da and an isoelectric point of 7.71.Multiple sequence alignment revealed that Cfi proteins were well conserved with the typical modular architecture and identical active sites throughout the vertebrates, included mammal, birds, cartilaginous fish, bony fish and amphibians Cfi, and the results showed that the sequences similarity wereamong 39%- 65%, which suggested the conserved function of Cfi. Phylogenetic analysis indicated that Cs Cfi and the homologous Cfi sequences from teleosts clustered into a clade, separating from another clade from the cartilaginous fish and other vertebrates. A signal peptide was defined at N-terminus, resulting in a 626-residue mature protein.The predicted Cs Cfi amimo acid sequence contained five domains: FIMAC, SRCR, LDLa1, LDLa2 and Tryp-SPc. Cs Cfi had a cleavage site of four amino acids Arg-Val-Arg-Arg（RVRR） between heavy chain and light chain, and only the Tryp-SPc domain in the L-chain.The glycosylation pattern of Cs Cfi containing 7 N-linked oligosaccharides is roughly identical toother teleosts, and six of them was located in H-chain. There are also amount of conserved Cys amino acids in Cfi amino acids. 1.2. Identified and function analyzied of recombinant protein of CfiAccordingto the validated sequence of Cs Cfi, specific primers was been designed to amplified the open reading frame sequence of Cs Cfi from the template of liver c DNA, and bulided prokaryotic expression vector with p EASY-E1 vector followed the principle of T-A cloned. After that, it would betransformed into BL21（DE3）ply Sand induced recombinant protein of Cs Cfi（r Cs Cfi）.The mature peptideof Cs Cfi would be separated and purifiedby AKTApurifer Box-900（Amersham Biosciences of GEHealthcare） with the His Trap TM FF crude column（5 m L） according to the manufacturer’s instruction.The resultant protein was boiled for 5 min, analyzed through 12.0% sodium dodecyl sulfatepolyacrylamide gel electrophoresis（SDS-PAGE） and visualized with Coomassie brilliant blue R-250.Weidentified the purified r Cs Cfiprotein with western blot and mass spectrometrytechnologies,western blotresult showed that a blot band at the corresponding location（approximately 71 k Da） of r Cs Cfi, suggesting specificity for this recombinant protein, and mass spectrometryresults showed that the nominal mass（Mr） of fusion protein was found to be 71.38 k Dawith the calculated isoelectric point value 6.67 and fifteen peptide fragments of r Cs Cfi protein with 62% sequence coverage rate of the deduced amino acid sequence. The plate count method was carried out to detect the antimicrobial activities against the Gram-positive bacteria Staphylococcus aureus and the Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa and Shewanella putrefaciens.The results showed thatr Cs Cfi protein were broad-spectruminhibitory effect, the minimal inhibitory concentration（MIC） to E. coli was between 0.03 mg/m L and 0.08 mg/m L, and the MIC to S. aureus, P. aeruginosa and S. putrefaciens were between 0.01 mg/m L and 0.05 mg/m L.In addition, after injection with active r Cs Cfi protein from the concentrations of 0.6 μg/g, 0.8 μg/g, 1.0 μg/g, 1.2 μg/g and 1.5 μg/g, and four tissues including liver, spleen, gut and head kidney were collected from each individual at five time-points（0 h, 6 h, 12 h, 24 h and 48 h） post injection to detected the relative expression of Cs Cfh and Cs C3-1. The results showed that the two genes in different tissues revealed different expression patterns post injection and showed an analogous fluctuation underthe five different concentrations and the relative expression quantities of Cs Cfh and Cs C3-1were equilibrium in all the tissues and could be induced of high transcript levels in liver, spleen and gut with 1.5 μg/g.Through pre-experiment, protein with the ideal concentration of 1.5μg/g was finally selected to develop a formal experiment. The method used was the same with that for pre-experiment. Three individuals were randomly sampled at 7 time-points of 0 h, 12 h, 24 h, 36 h, 48 h, 72 h, and 96 h, and the results showed Cs Cfh and Cs C3-1 transcript levels exhibited distinct time-dependent response patterns in head kidney, spleen, gut and liver. The maximum of spleen, liver and gut were appeared at 6h, 12 h and 48 h, respectively. In kidney, the sxpression of Cs Cfh and Cs C3-1 were down-regulated expression,and achieve the lowest point at 24 h post fection. The research revealed Cs Cfi plays an important role in C. Semilaevis immunity. 2.Preliminary study of Cs C3-1 2.1 Indetified andsequence analysisof Cs C3-1According to the builted genome-wide c DNA library of our laboratory from half smooth tongue sole, the clone number is csgtsa0₀003_A04.The full-length c DNA of Cs C3 was acquired by use of rapid amplification c DNA end technology, it was contain 5130 bp in length, including a 37 bp 5’- untranslated region（UTR）, a 128 bp 3’-UTR and a 4965 bp open reading frame（ORF）. It encoded a polypeptide of 1655 amino acids, with a molecular mass of 184.98 k Da and an isoelectric point of 6.46. The predicted Cs C3-1 amimo acid sequence contained signal peptide（1-23 aa, SP）, alpha-macroglobulins domain（130-227 aa, A2M-N）, alpha-macroglobulins domian（453-593 aa, A2M-N-2）, allergy toxin homologous structure domain（682-717 aa, ANATO）, alpha-macroglobulins domain（759-858 aa, A2M）, alpha-macroglobulins thiol lipid formation structural domain（992-1022 aa, Thiol-ester-C1）, alpha-macroglobulins complement component domain（1044-1274 aa, A2M-Comp）, alpha-macroglobulins recepter domain（1044-1274 aa, A2M-recep）, and nerve growth factor the c-terminal domain structure（1516-1636 aa, C345C）.Phylogenetic analysis indicated that Cs C3-1 and the homologous C3-1 sequences from teleosts clustered into a clade, separating from another clade from the mammaland other vertebrates.Multiple sequence alignment and showed thethe sequences similarity wereamong40%- 69%, which suggested the conserved function of structure of complement C3-1 was conserved though evolution. 2.1 Transcript profiles of before and after challenged with Vibro anguillarumAccording to themedial lethal concentration of Vibro anguillarum from half smooth tongue in our laboratory, 2×107 colony forming units（CFU） of V.anguillarumwere intraperitoneally injected into each challenged fish to study the relative expression of Cs C3-1, we identified the expression pattern in 9 normal tissueses（Liver, Spleen, Head Kid, Blood, Skin, Brain, Muscle, Gill and Gut） and 4 tissues（liver, spleen, kidney and blood） in several time-points post challenged with Vibro anguillarum. RT-PCR results showed C3-1 was transcript in all the tissues which has been detected, it was predomainly espressed in brain, intermediately expressed in liver, muscle and blood, and slightly in kidney and gill, the express quantity in liver and brain was higher than others significantly. Significant up-regulations or down-regulations were observed in all the detected tissuesafter infection with Vibro anguillarum. In the liver and blood, the maximum expressive peaks were observed at 12 h, 5.5 times and 15.5 times before challenge, respectively. After that, it was appeared down-reguantions in liver and minimum expressive quantity was observed at 24 h, 0.3 times before infect. In head kidney, the relative expression of C3-1 was reached a higher quantity change at 6h and 24 h, and reached its peak at 24 h, dozens of times before infect. In spleen, the relative expression of C3-1 was emerged down-regulatons at all the time-points, and return to the initiate expression of 0h.In this study, considerable efforts had been made to acquire the full length of c DNA and animo acid sequencesof Cs Cfi and Cc C3-1, and demonstrated the antimicrobial activities and the function that could induce different expression patterns of Cs Cfh and Cs C3-1 of r Cs Cfi protein. Finally, Cs C3-1gene showed specific expression patterns post injected with V. anguillarumfrom differences tissues. From a biological perspective of this study,a referential orientation for the aquaculture science develop of C. semilaevis has been provided. The development of antibacterial products and the application of feed additives were significative for healthful aquaculture and food security.