Chemokines are cytokines that can promote the activation and migration of immune cells,and increase the recognition of antigen by antigen-presenting cells(APC).Previous studies showed that a DNA vaccine can induce humoral and cellular immune responses of flounder after immunization.To explore the improvement of chemokines on the efficiency of OmpK vaccine,two bicistronic DNA candidate vaccines were constructed and the immune responses they induced in the flounder were investigated by reverse transcription polymerase chain reaction(RT-PCR),indirect immunofluorescent assay(IFA),H&E staining,flow cytometry(FCM),and quantificational real-time polymerase chain reaction(qRT-PCR).pBudCE4.1 plasmid as an expression vector,bicistronic DNA vaccines encoding OmpK gene and CC-motif ligand 4 gene(p-OmpK-CCL4),or Ompk gene and CC-motif ligand 19 gene(p-OmpK-CCL19)were successfully constructed.The results showed that two bicistronic DNA vaccines expressed Ompk protein of Vibrio anguillarum and CCL4/CCL19 proteins of flounder both in vitro and in vivo.After immunization,a large number of leucocytes in muscle were recruited at the injection site in treatment groups.The constructed vaccines induced significant increases in CD4-1+and CD4-2+T lymphocytes,and sIgM+B lymphocytes in peripheral blood,spleen,and head kidney.The percentage of T lymphocytes peaked on the 14th post-vaccination day whereas that of B lymphocytes peaked in the 6th post-vaccination week.Moreover,the expression profiles of 10 immune-related genes increased in muscles around the injection site,spleen,and head kidney.After the challenge,p-OmpK-CCL4 and p-OmpK-CCL19 conferred a relative percentage survival(RPS)of 74.1％and 63.3％,respectively,higher than p-OmpK alone(40.8％).In conclusion,both CCL4 and CCL19 can improve the protection of p-OmpK via evoking local immune response and then humoral and cellular immunity.CCL4 and CCL19 will be potential molecular adjuvants for use in DNA vaccines.

Hanlin LI;Jing XING;Xiaoqian TANG;Xiuzhen SHENG;Heng CHI;Wenbin ZHAN

Laboratory of Pathology and Immunology of Aquatic Animals,KLMME,Ocean University of China,Qingdao 266003,China;Laboratory for Marine Fisheries Science and Food Production Processes,Pilot National Laboratory for Marine Science and Technology(Qingdao),Qingdao 266071,China

海洋湖沼学报（英文版）

[1]Hanlin LI;Jing XING;Xiaoqian TANG;Xiuzhen SHENG;Heng CHI;Wenbin ZHAN-.Two bicistronic DNA vaccines against Vibrio anguillarum and the immune effects on flounder Paralichthys olivaceus)[J].海洋湖沼学报（英文版）,2022(02):786-804

flounder,Chemokines,OmpK,pBudCE4,Ompk,1+and,2+T,sIgM+B

Two,bicistronic,vaccines,against,Vibrio,anguillarum,immune,effects,Paralichthys,olivaceus,are,cytokines,that,promote,activation,migration,cells,recognition,antigen,by,presenting,APC,Previous,studies,showed,humoral,cellular,responses,after,immunization,To,explore,improvement,chemokines,efficiency,two,candidate,were,constructed,they,induced,investigated,reverse,transcription,polymerase,chain,reaction,indirect,immunofluorescent,assay,IFA,staining,flow,cytometry,FCM,quantificational,real,qRT,plasmid,expression,vector,encoding,motif,ligand,CCL4,CCL19,successfully,results,expressed,proteins,both,vitro,vivo,After,large,number,leucocytes,recruited,injection,site,treatment,groups,significant,increases,CD4,lymphocytes,peripheral,blood,spleen,head,kidney,percentage,peaked,14th,post,vaccination,day,whereas,6th,week,Moreover,profiles,related,genes,increased,muscles,around,challenge,conferred,relative,survival,RPS,respectively,higher,than,alone,In,conclusion,protection,via,evoking,local,then,immunity,will,potential,molecular,adjuvants,use

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