Characteristics Of Intestinal Microflora And C9 Gene From Cynoglossus Semilaevis Inimmune Response To Vibrio Anguillarum Infection

In this study,the bacterial community's structures and their correlativity in half-smooth tongue sole(Cynoglossus semilaevis)in individuals with different immunity states were analyzed with the help of 16S rRNA sequence.One library was prepared under normal conditions(control,CG);two libraries were prepared during Vibrio anguillarum infection,where vibriosis symptoms were obvious and non-obvious(VAHG and VANG,respectively;two were prepared during normal cultivation conditions,fish with or without any symptoms in the same pool(HOSG and NOSG,respectively)were analyzed using high-through sequencing technique.In total,150 million reads and 27,475 OTUs were obtained,which belonged to 58 phyla,154 class,241 order,313 family and 609 genera.In total,21 bacterial phyla were shared by all groups.The KEGG pathway analysis showed that all the bacteria in the gut of C.semilaevis had participated in 123 different pathways.A pathway which is called Bacterial secretion system(belonged to Membrane transport)was enriched in HOSG group when compared with pathways in all the other groups.These bacteria may be important pathogen which leads to these diseases.The analysis of intestinal microbial macro genome showed that complement C9 gene expression was high after the pathogen stimulation in C.Semilaevis.In the present study,a truncated C9 cDNA sequence encoding 516 amino acids was cloned and characterized in the C.Semilaevis,this truncated C9 presents TSP1,LDLRA and MACPF domains.The full-length cDNA of CsC9 was 2148bp in length,including a 1548bp open reading frame(ORF).It encoded a molecular mass of 57.12 kDa and an isoelectric point of 5.65.Multiple sequence alignment revealed that the amino acid sequence of CsC9 shared the highest similarities with C9 sequences from Paralichthys olivaceus.RT-PCR analysis detected these C9 transcripts demonstrating constitutively expressed in all tested tissues with the predominant expression in liver and the lowest expression in brain.Temporal expression levels of CsC9 after challenging with Vibrioanguillarum showed different expression patterns in intestine,spleen,gill,head kidney and liver.In addition,the recombinant CsC9(rCsC9)protein was achieved and analysed using 2-dimensional gel electrophoresis.Temporal expression levels of CD59,CR1 and CR2in lymphocytes with recombinant C9 protein infected showed a rising trend.These results indicate that CsC9 might important role in immune response of C.Semilaevis against bacterial infection.