Paralichthys Olivaceus Ly-6D Gene Expression And Function Analysis

Paralichthys olivaceus is an extremely important economic fish in China.With the rapid development of economy,the production of flounder is increasing,and the disease problem is also aggravated.Lymphocyte antigen 6(Ly6)family members are involved in both innate and acquired immunity and play an important role in the immune system.Due to the low homology among members of the Ly6 family,its functions are also different.The function of Ly-6D,a core member of the Ly6 family in teleost is completely unknown.In this paper,Ly-6D(PoLy-6D)of Paralichthys olivaceus was cloned and identified,and its biological functions were studied.Sequence analysis results showed that the open reading frame(ORF)of PoLy-6D was 300 bp and encoded 99 amino acids,among which the first 19 amino acids were signal peptide sequences.PoLy-6D lacks the glycosylphosphatidyl inositol(GPI)anchoring structure and is predicted to be a secreted protein.The three-dimensional structural analysis shows that the spatial structure of PoLy-6D is similar to that of human CD59,both of which are three-fingered structures composed of three β folding sheets and an α helix.These results suggest that PoLy-6D and CD59 may have some similar functions.In order to further explore the function of PoLy-6D,we carried out in vitro prokaryotic expression.The results showed that the recombinant protein PoLy-6D(r PoLy-6D)inhibited the cleavage of rabbit erythrocytes by Japanese flounders serum.And it may inhibit the activation of complement by Japanese flounders serum.At the same time,we found that r PoLy-6D can reduce the bactericidal activity of serum,but the effect of r PoLy-6D on the bactericidal activity of serum is changed for different bacteria.Our results showed that r PoLy-6D can significantly reduce the bactericidal activity of serum on opportunistic pathogens such as Micrococcus luteus and Pseudomonas fluorescens,but had no significant effect on E.piscicida.Moreover,the survival rate of PoLy-6D in serum was significantly reduced after blocking.These results indicate that PoLy-6D plays a role in regulating complement.In addition,we found that r PoLy-6D can also bind to a variety of bacteria,but it does not have antimicrobial activity,which indicates that PoLy-6D may act as a pathogen receptor to participate in pathogen clearance by the host.Next,we analyzed the expression profile of PoLy-6D in flounder.Tissue specific test results showed that PoLy-6D was expressed in 8 tissues(muscle,blood,spleen,head kidney,gill,liver,heart and intestine)of healthy flounder,and the highest expression level was found in spleen,followed by head kidney,and the lowest expression level was found in intestine.When the intracellular pathogen Edwardsiella piscicida was used to infection,the expression of PoLy-6D tended to be up-regulated,but when the extracellular pathogen Vibrio anguillarum and infectious spleen and kidney necrosis virus(ISKNV)were used to infection,the expression of PoLy-6D in liver,spleen and head kidney tended to be down-regulated.Under different microbial infection,the expression levels of PoLy-6D in different immune tissues changed,suggesting that PoLy-6D may be involved in the body’s immunity against disease infection.In order to further explore whether PoLy-6D participates in pathogen clearance by the host in vivo.We constructed an in vivo overexpression vector.After it confirms that PoLy-6D was overexpressed in flounder,E.piscicida was used to infect the flounder.And the experimental results showed that the number of bacteria in immune organs of overexpressed PoLy-6D decreased significantly.These results suggested that the overexpression of PoLy-6D could enhance the host’s resistance to E.piscicida,indicating that PoLy-6D was associated with the immune response process of flounder.In conclusion,this study found that PoLy-6D is a novel complement inhibitor.Overexpression of PoLy-6D in vivo can improve host resistance to pathogen infection,and PoLy-6D has antibacterial immunity in flounder.PoLy-6D can bind to both Gram-positive and Gram-negative bacteria,and may play a role as pathogen recognition receptor.These results not only provide a theoretical basis for its application in fishery vaccine adjuvant,but also provide a new insight for the regulation mechanism of complement system in bony fish and provide new ideas for healthy and efficient culture of flounder.