Study Of Functional Characteristics And Immunoprotecive Of ?-enolase Of Streptococcus Iniae In Channel Catfish(Ictalurus Punctatus)

Streptococcus iniae is a ?-hemolytic streptococcus,it has two serotypes?I and II?and does not belong to any type of Lancefield.S.iniae was first reported on dolphin from Amazon basin,and it spread all over the world,such as Israel,Australia,Japan and the United States,which caused serious economic losses to global aquaculture and has been regarded as one of the strongest pathogens in aquaculture.S.iniae has a broad host,kinds of cold-water fish and warm-water fish are susceptible,such as rainbow trout,tilapia,salmon,barramundi,yellow tail halibut and sea bass,it also does great harm to other animals and people which are old or immunocompromised,causing widespread attention.However,the study on pathogenic mechanism and vaccine of S.iniae is still in its initial stage.This study focused on the a-enolase,an important virulence factor of S.iniae,to investigate the potential role during the infection and the cross-protection;In addition,the protective effect of vaccine combinated IFN-? adjuvant was studied and its potential immunological mechanism was explored in channel catfish.The purpose of this study is to improve the immune effect of the vaccine and to provide a new idea for the immune control of S.iniae disease.1.Cloning,expression and biological functions analysis of a-enolase of S.iniaeThe sequence of a-enolase was amplified based on the genomic DNA of Streptococcus iniae DGX07.The bioinformatics analysis showed that:the full ORF of ?-enolase?Accession No.AGT63054?was 1308bp,encoding 435 amino acids and the protein was predicted as 47.24 KDa.The amino acid sequence of S.iniae had a high identity comparing with it in others streptococcus through Blastp analysis in NCBI.The active sites including:two enzyme active sites?205 E,343 K?,three metal binding sites?242 D,291 E,318 D;magnesium?,ten substrate binding sites?155 H,164 E,291 E,318D,343 K,370-373 SHRS,394 K?and one plasminogen binding sites?248-256 FYDKERKVY?.A number of B cell epitopes also were predicted and there was no signal peptide cutting sites and transmembrane regions.According to the phylogenetic tree we found that the amino acid sequences of enolase in prokaryote and eucaryon distributed in different two clusters,for example the sequences of human,mice,cattle,chicken and zebrafish grouped in one cluster,which far away from in evolutionary relationships comparing with prokaryote.And in prokaryote,all streptococcus gathered in one cluster.showing a close genetic evolution.These results showed that the gene structure and functional active sites of ?-enolase of S.iniae consisted with enolase.The recombined a-enolase?rENO?was obtained by prokaryotic expression,and the biological functions of which has also been tested.The location of a-enolase in S.iniae was confirmed by the rabbit anti-rENO serum,and it showed that the a-enolase not only existed in cytoplasm but also the surface,even out of the bacteria.The interaction of a-enolase and plasminogen?Pig?was performed by the reaction of rabbit anti-rENO serum and rabbit anti human plasminogen antibodies,which showed that the recombined rENO can also bind the human Pig like the natural a-enolase does.Using rabbit anti-rENO serum blocking the a-enolase on S.iniae,caused the adhesion?60.39%of control group?and invasion?47.76%of control group?to EPC cells both declined.And it also could catalyze 2-PGE transform into PEP,like enolase.These results showed that the a-enolase of S.iniae not only existed in cytoplasm,but also located on the surface of bacteria,whilst possessed moonlighting functions.2.Cross-protective of recombinational a-enolase?rENO?of S.iniae in channel catfishThe genome DNA of S.iniae DGX07,ATCC29177 and ATCC29178 were performed by RAPD analysis using random primer.The results showed that three strains were amplified multiple DNA fragments between 750-2000 bp.DGX07 and ATCC29178?serotype ??were both amplified a fragment about 750 bp,but not showed in ATCC29177?serotype ??,therefore,the DGX07 belonged to serotype I S.iniae.Multiple alignment analysis of amino acid sequence of a-enolase showed DGX07 had identity and similarity above 99%with different serotype S.iniae strains and S.agalactiae?96.6%and 98.2%,respectively?,but has low identity and similarity with Edwardsiella ictaluri,Aeromonas hydrophila and Yersinia ruckeri?all just more than 50%?.Western-blot showed that rabbit anti-rENO serum reacted with a-enolase of different serotype S.iniae and S.agalactiae,but none of E.ictaluri,A.hydrophila and Y.ruckeri.The immune protective effect showed that antibody level of vaccinated group increases at 7 d,reached peak at 14 d,declined at 28 d,but still keep relative high level.However,antibody level of vaccinated group was not significant compared with control group at 56 d?p<0.05?.Cross-protective effect of rENO showed that the relative protective survival?RPS?was 45.0%in vaccinated group by rENO when challenged with serotype I S.iniae?DGX07?,and 44.4%RPS in vaccinated group by rENO when challenged with serotype ? S.iniae?ATCC29177?.These results indicated that a-enolase was high conservative in Streptococcus and had cross-protective to against serotype ? and type ? of S.iniae.3.Construction and evaluation of protective effect of adjuvant vaccine combined recombinant IFN-? of channel catfish and rENO in channel catfishThe IFN-y gene was amplified based on the cDNA of head kidney of channel catfish.The bioinformatics analysis showed that:the full ORF of IFN-? was 528 bp,encoding 175 amino acids and the length of protein was predicted as 20.74 KDa:one signal peptide cutting site was found between number 20 and 21 amino acids and there was no transmembrane region.After BLASTP in NCBI,the results showed that the cloned sequence of IFN-y had a high identity/similarity compared with IFN-?2a?NP₀01187231.1??98.3%/98.3%?and IFN-?2b?NP-001187263.1??100%/100%?,while there were only relative low identity/similarity compared with IFN-yl?NP₀01187146.1??19.1%/38.7%?.The phylogenetic tree showed that the IFN-? of teleost grouped in one cluster,and the IFN-y of others vertebrates grouped in another cluster.The cloned IFN-y got together with the IFN-y2a and IFN-?2b,considered with the high identity/similarity the cloned IFN-? was recognized as IFN-?2b of channel catfish.Fusion protein rIFN-y?about 38 KDa?were expressed in prokaryotic expression system using the mature peptide of IFN-y?about 18.17 KDa?.The macrophages were isolated from head kidney of channel catfish and stimulated with rIFN-?,whilst the expression of immune-related genes was analyzed by real-time PCR.The results showed that the expression of IL-1?1,TNF-?,MHCII and STAT1?p<0.05?were significantly up-regulated after 24h post-stimulation,and the expression of NOS2b was only a small increase.Moreover,there was no significant change in negative group and pET32a fusion tag protein group?p>0.05?These results indicated that cloned IFN-y2b of channel catfish had immunomodulatory properties and could be used as and cytokine adjuvant in vaccine.The channel catfish were immunized with rIFN-? combined with rENO and inactivated S.iniae,respectively,and the commercial fish adjuvant MontanideTM ISA 763 AVG as control.The immune index analysis showed that the activity of ACH50 and lysozyme can be induced by rIFN-? and MontanideTM ISA 763 AVG.However,the induce features were different,the enhance effect of rIFN-? was observed in initial phase?before 28 days post vaccination,dpv?,but the activity of ACH50 and lysozyme were maintained in a high level and a long time?56 dpv?in MontanideTM ISA 763 AVG stimulated group.Moreover,the MontanideTM ISA 763 AVG significantly increased the level of specific antibodies?p<0.05?,but the rIFN-? could not?p>0.05?.The expression of IL-1 ?1,TNF-?,MHCII and STAT1 were up-regulated in initial stage?before 14 dpv?stimulated by rIFN-?,but the expression of NOS2b was down-regulated.The MontanideTM ISA 763 AVG could induced these genes up-regulation at 3 dpv to 30 dpv.The RPS of MontanideTM ISA 763 AVG,rIFN-?,rENO,inactivated vaccine,rIFN-?+ rENO?rIFN-? + inactivated vaccine,MontanideTM ISA 763 AVG+ inactivated vaccine and MontanideTM ISA 763 AVG+rENO in the fourth week and the eighth week were 5%,0%,40%,65%,60%,75%,90%,75%and 0%,0%,10%,20%,10%,15%,40%,35%,respectively.These results indicated that both of rIFN-? and MontanideTM ISA 763 AVG could enhance the activity of ACH50 and lysozyme,induce specific antibodis and expression of immune relative genes,and increased immune protective rate.However,rIFN-y mainly induced the early immune response and MontanideTM ISA 763 AVG mainly induced the later immune response in channel catfish.4.Construction and evaluation of protective effect of fusion DNA vaccine with S.iniae a-enolase and channel catfish IFN-? in channel catfish.The gene fusion of IFN-? and a-enolase was performed by overlap extension PCR,and two flexible linkers?Gly Gly Gly Gly Ser?2 were added in the joint.The eukaryotic expression vectors of pcDNA3.1-ENO,pcDNA3.1-IFN-? and pcDNA3.1-IFN-y-ENO were obtained by cloning.All the plasmids were transfected into EPC cells and detected by immunofluorescence.Results showed that green fluorescent signals were observed in pcDNA3.1-IFN-?,pcDNA3.1-ENO and pcDNA3.1-IFN-?-ENO group,but no signal in pcDNA3.1 and negative control group,which demonstrated that all the plasmids expressed in EPC cells.All the plasmids were injected in channel catfish by intramuscular injection?i.m.?and the immunohistochemical analysis was performed with tissues of muscle and kidney at 14 days post injection.The positive signals were detected in muscle cells and kidney macrophages,but there was no signal in control group,which indicated that the recombinant vector expressed in channel catfish.The immune index analysis showed that the fusion DNA vaccine of pcDNA3.1-IFN-?-ENO could significantly increase the activity of ACH50 and lysozyme?p<0.05?,which were maintained a high level and a long time?56 dpv?.While IFN-y could significantly enhance the antibody level of fusion DNA vaccine?p<0.05?.The expression of IL-1?1,TNF-?,MHCII,STAT1 and NOS2b were all up-regulated after 7 dpv by pcDNA3.1-IFN-?,pcDNA3.1-ENO and pcDNA3.1-IFN-?-ENO,and reached to the peak at 14 dpv or 28 dpv.However,the expression of NOS2b wad down-regulated after 48 hours post challenge.The RPS of pcDNA3.1,pcDNA3.1-IFN-?,pcDNA3.1-ENO and pcDNA3.1-IFN-?-ENO in the fourth week and the eighth week were 0%,15%,75%,85%and 0%,0%,45%,60%,respectively.These results indicated that fusion DNA vaccine of pcDNA3.1-IFN-?-ENO could enhance the innate immune response,induce generation of specific antibodis and expression of IL-1?1?TNF-??MHCII?STAT1 and NOS2b gene,increase immune protective rate,and had a longer immune protection effect.Therefore,it has the potential to develop as an economical and effective adjuvant vaccine.