Molecular Cloning Of Red Snapper IL-6 And Preliminary Study On Its Adjuvants Immunocity On Vibrio Harveyi OmpW DNA Vaccine

Red snapper(Lutjanus sanguineus)is widely distributed in the coastal areas of Red Sea,the eastern coast of Africa,Philippines and Japan.It is one of the economically important fish in the South coastal regions of China,such as Taiwan Strait,QiongZhou Strait,Hainan Island,and Beibu Gulf.In recent years,with the increasing density of marine fish farming,red snapper has burst massives of bacterial diseases and caused huge economic losses.The surface unique outer membrane protein of Vibrio harveyi is playing an important role in stabilizing the structure of the bacterial cells,the exchange of substances between cells and pathogenic bacteria processes.As a key component of vaccine,outer membrane protein has a good immunogenicity,which can stimulate to produce a variety of immune responses.Interleukins is an important cytokines,which can expand and regulate a variety of immune response in cell-cell interactions.While some cytokine can be used as a vaccine molecule immunoadjuvant to enhance DNA vaccine effect.The full-length cDNA of red snapper IL-6 was cloned using homological cloning and rapid amplification of cDNA ends(RACE)methods.Results showed that the full-length of IL-6 cDNA was 1307 bp,containing an open reading frame of 639 bp encoding 212 amino acids with an estimated molecular weight of 24.0 kDa and an estimated isoelectric point of 5.65.Amino acid homology analysis revealed that the red snapper IL-6 amino acid share a homology of 53%with the Epinephelus coioides IL-6.Red snapper IL-6 genomes was 3447 bp,containing five exons and four introns.The expression pattern of IL-6 in head kidney,thymus,spleen,liver,gill,intestine and muscle of the fish induced by Vibrio harveyi infection was analyzed by fluorescent quantitative real-time PCR technology.The results showed that the expression of IL-6 mRNA could be detected in head kidney,thymus,spleen,and the highest expression occurred in 6-18 h post infection.Partial IL-6 which is dislodged the signal peptide fragments was cubcloned into prokaryotic expression pET-32a(+)vector.The recombinant IL-6 fusion proteins were overexpressed in E.coli BL21(DE3)cells in the presence of isopropyl-P-thiogalactopyranoside(IPTG).The recombinant IL-6 fusion proteins were purified by His TrapTM HP column,and then identified by western-blot using His-Tag Mouse mAb.Polyclonal antibody against IL-6 fusion protein was raised in Kunming-mice immunized with the purified IL-6 fusion protein,and the antibodytiter reached 1:40000.Western-blot analysis found that the specific polyclonal antibody can react with the recombinant IL-6 fusion protein,head kidey,thymus and spleen.Immunohistochemistry analysis showed that IL-6 proteins mainly distributed in the cytoplasm of head kidey,thymus and spleen.A variety of outer membrane proteins of Vibrio harveyi have good immunogenicity.While,we found the immunogenicity of OmpW is better than the other bacteria's outer membrane protein.Therefore,we first fuse the red snappers IL-6 and Vibrio harveyi OmpW using the overlapping extension technology.The chimeric gene is 1263 bp with an estimated molecular weight of 46.2 kDa.Then the chimeric gene was cloned into the prokaryotic expression vector pET-32a(+)to construct recombinant plasmids.Western-blot analysis indicated that specific antigen-antibody reaction was occurred between the antiserum and its corresponding recombinant fusion protein.In order to understand the role of IL-6 as a DNA vaccine molecular adjuvant,we subclone IL-6,OmpW and chimeric gene IL6-OmpW into the eukaryotic expression vector pcDNA3.1(?).Lutjanus sanguineus was intramuscular injected with the three plasmids.The effect of DNA vaccines were evaluated on nucleic acid and protein levels.The investigation on distribution and expression of the three plasmids indicated that the recombinant plasmid DNA is distributed in muscle,spleen,head kidney,liver 7 days and 28 days after injection.Results of RT-PCR and PCR indicated that the three genes expressed in above all tissues of the fish on 7th day and 28th day of injection.Indirect ELISA and Western-blot analyses revealed that the target protein was expressed to activate antibody reponse in vivo.Challenging experiment with live bacteria,the RPS of recombinant plasmid pcDNA-IL6,pcDNA-OmpW and pcDNA-IL6-OmpW are 36%,60%and 76%.The result showed that the three plasmids had immune protective effect with fish,and also proved IL-6 as a molecular adjuvant which could enhance immune protective effect.