Cloning, Expression And Effects Of Vaccine Adjuvant Of Ictalurus Punctatus Channel Catfish IL-8

Fishery vaccines play an important role in the control of aquatic animal diseases, meanwhile avoiding contamination of antibiotics and other drugs on the water environment and aquatic products. Adjuvant as a group of structurally heterogeneous compounds is able to modulate the intrinsic immunogenicity of an antigen. Cytokines, produced by the animal’s own immune cells and other correlative cells in the use of adjuvants are more advantageous than conventional adjuvants. Interleukin 8 (IL-8) is a CXC chemokine produced by numerous cell types. In mammals, it is known to be produced by macrophages/monocytes, epithelial cells, neutrophils, fibroblasts, and endothelial cells upon infection or stimulated by cytokines such as IL-1β and TNF-a. Being a CXC chemokine, IL-8 predominantly promotes the recruitment of neutrophils and has been shown to chemoattract T lymphocytes and alter cytokine production from T cells. IL-8 is also a chemoattractant for other cell types such as basophils, T lymphocytes, and NK cells, and also enhances permeability of endothelial cells. In mammals and fish, some studies have focused on the potential use of IL-8 as an adjuvant for DNA vaccination. In this paper, it was investigated that whether channel catfish (Ictalurus punctatus) IL-8 (CcIL-8), as immune adjuvant, could enhance the protective immunity to the Streptococcus iniae (S.iniae) vaccine, including:1 doing and sequence analysis of the channel catfish IL-8An IL-8 gene was amplified from the spleen of channel catfish using RT-PCR and was cloned into the vector pMD19-T to construct the recombinant plasmid. The nucleotide sequence of the amplified IL-8 gene and the amino acid sequence deduced from the gene were then analyzed by the bioinformatics tools and online server. The results showed that IL-8 nucleotide sequence contained a complete opening reading frame which was 336 bp (GenBank accession number KP701473) and encoded 111 amino acid residues. The protein had a conserved chemokine_CXC domains, five cysteine residues, the signal peptide and the transmembrane regions. Also it possessed many important sites related to post-translational modification including 2 potential phosphorylation sites and 2 potential N-glycosylation sites. Its secondary structure was composed by alpha-helix and extended strand. This study got the short spliced transcripts of IL-8 in the channel catfish and its deduced amino acid sequence shared a high homology with IL-8 of Heteropneustes fossilis, along with sequences identities 87.1% and the same cluster on the phylogenetic trees, but less related to other teleost fish, such as carp, zebrafish and rainbow trout.2 CcIL-8 mature peptide gene prokaryotic expression and effects of S.iniae subunit vaccine as adjuvantAccording to the cloned CcIL-8 gene without signal peptide, we designed one pair specificity primer to amplify the mature peptide (mCcIL-8). It was obtained a gene of the length of 264 bp. Then the mCcIL-8 gene was cloned into pET-32a(+) vector, and prokaryotic expression vector pET-32a(+)-mCcIL-8 plasmid was constructed. Transforming into prokaryotic expression strain BL21(DE3), a protein of 29kDa was expressed. After renaturation by Ni-NTA affinity chromatography, dilution and dialysis, Werstern bolt and SDS-PAGE results showed purified recombinant fusion protein was expressed and purified successfully. To investigate the immunoenhancement of renatured recombination mCcIL-8 (rmCcIL-8) protein with the rENO protein-based S.iniae subunit vaccine (S.iniae-rENO), rmCcIL-8 protein (20 μg/fish) with S.iniae-rENO (50μg/fish) was co-inoculated to channel catfish (80±10 g) by intraperitoneal injection, the control groups including rmCcIL-8 protein adjuvant group, S.iniae-rENO subunit vaccine group and PBS group. (1) Innate immunity:collecting blood serum at 1,3,7,14,21,28 and 56 d post immunization measured lysozyme and ACH50 activity. Results:S. iniae-rENO+rmCclL-8 group lysozyme activity was significantly different (P<0.01) than S:iniae-rENO group at 1, 3 and 7 d post immunization, and there was no significant difference between vaccine groups at 14 d post immunization, but was significantly different (P<0.01) than rmCcIL-8 group and PBS group. There was no significant difference between groups at 21 and 56 d post immunization. S.iniae-rENO+rmCcIL-8 group ACH50 activity was different (P<0.05) than S.iniae-rENO group at 1 and 3 d post immunization, and there was no significant difference between vaccine groups at 7 and 14 d post immunization, but was significantly different (P<0.01) than rmCcIL-8 group and PBS group. There was no significant difference between groups at 21 and 56 d post immunization. Showing rmCcIL-8 protein could enhance the subunit vaccine S. iniae-rENO induced lysozyme and ACH50 level within a certain time. (2) Serum antibody ELISA:collecting blood serum at 7,14,21,28,35,42 and 56 d post immunization detected rENO-specific serum antibodies by indirect ELISA. Results:vaccine groups had detected antibody at 14 d post immunization. S.iniae-rEN0+rmCcIL-8 group antibody level was different (P<0.05) than S.iniae-rENO group at 14 d to 42 d post immunization, reaching a peak at 28 d post immunization, decreasing at 35 d post immunization, and were unable to detect antibody at 56 d post immunization. Showing rmCcIL-8 protein could increase antibody levels with the subunit vaccine S.iniae-rENO, but failed to extend the duration of antibody production. (3) Expression of immune-related genes:head kidney was taken from the fish (vaccine groups and PBS group) at 24 h post the first chanllenge (at 28 d post immunization) to detect the expression immune-related genes by qRT-PCR. Results:S.iniae-rENO+rmCcIL-8 group the relative expression of IL-1β,TNF-α, CXCL10 and MHCⅡβ were different (P<0.05) than S.iniae-rENO group. Showing the subunit vaccine S.iniae-rENO+rmCcIL-8 trigger a stronger immune response and MHCⅡ antigen-presenting. (4) Protective efficacy of challenging:At 28 and 56 d post immunization, fish were chanllenged with S.iniae DGX07 respectively. Results:the first challenge, S.iniae-iENO+rmCcIL-8 group (71.42%) had higher RPS than S.iniae-rENO group (49.99%), but the two vaccines weren’t protective in the second challenge. Showing the subunit vaccine S.inae-rENO+rmCcIL-8 produced stronger protective immunity than the subunit vaccine S.iniae-rENO, and rmCcIL-8 protein couldn’t extend the protective effects.3 Construction of a recombinant eukaryotic expression plasmid containing MCcIL-8 gene and effects of S.iniae DNA vaccine as adjuvantThe gene CcIL-8 contained the Kozak and 6×CAC sequences (MCcIL-8) and cloned into eukaryotic expression vector pcDNA3.1(+) to generate the recombinant eukaryotic plasmid pcDNA3.1(+)-MCcIL-8 (pc-MCcIL-8). Transcription of MCcIL-8 gene was detected in muscle from channel catfish at 7,14,28 and 56 days post injection. In addition, at 14 days post injection production of MCcIL-8 protein was also detected in the muscle tissues of pc-MCcIL-8 injected fish by immunohistochemistry. To investigate the immunoenhancement of pc-MCcIL-8 plasmid DNA with the eno gene-based S.iniae DNA vaccine (pc-Sieno), pc-MCcIL-8 (25 μg/fish) with pc-Sieno (25 μg/fish) was co-vaccination to channel catfish (80±10 g) by intramuscular injection, the control groups including pc-MCcIL-8 gene adjuvant group, pc-Sieno DNA vaccine group and pcDNA3.1(+) empty vector (pc) group. (1) Innate immunity:collecting blood serum at 1,3, 7,14,21,28 and 56 d post vaccination measured lysozyme and ACH50 activity. Results: pc-Sieno+pc-MCcIL-8 group lysozyme activity was different (P<0.05) than pc-Sieno group at 3 to 56 d post vaccination, reaching a peak at 21 d post vaccination. ACH50 activity was significantly different (P<0.01) than pc-Sieno group at 3 to 56 d post vaccination, and reached a peak at 14 d post vaccination. Indicating pc-MCcIL-8 could significantly enhance the DNA vaccine pc-Sieno induced lysozyme and ACH50 level. (2) Serum antibody ELISA:collecting blood serum at 7,14,21,28,35,42 and 56 d post vaccination detected ENO-specific serum antibodies by indirect ELISA. Results: pc-Sieno+pc-MCcIL-8 group antibody level was significantly different (P<0.01) than pc-Sieno group at 14 to 56 d post vaccination, reaching a peak at 35 d post vaccination. Indicating pc-MCcIL-8 could significantly enhance the DNA vaccine pc-Sieno inducing antibody level. (3) Expression of immune-related genes:head kidney was taken from the fish (vaccine groups and pc group) at 24 h post the first challenge (at 28 d post immunization) to detect the expression immune-related genes by qRT-PCR. Results indicating the vaccine groups triggered a stronger antigen-presenting and immune response than pc group. pc-MCcIL-8 resulted in immunoregulation factor (IL-1β and TNF-a) secreting and MHCⅡntigen-presenting increasing. (4) Protective efficacy of challenging: At 28 and 56 d post immunization, fish were challenged with S.iniae DGX07 respectively. Results:the both of the challenges, pc-Sieno+pc-MCcIL-8 group (80% or 73.33%) had higher RPS than the pc-Sieno group (60% or 53.33%). Indicating the DNA vaccine pc-Sieno+pc-MCcIL-8 produced stronger protective immunity than the DNA vaccine pc-Sieno, and it could provide strong protective immunity against S.iniae about two months.