Molecular Cloning And Expression Of Non-specific Immune Genes From The Mandarin Fish, Siniperca Chuatsi

The mandarin fish (Siniperca chuatsi) is an important species in aquaculture industry in China. The understanding of its immune system is essential for its aquaculture. The present study aims to clone interleukin-8 (IL-8) and express five mandarin fish non-specific immune genes in Escherichia coli, including interleukin-1? (IL-1?), IL-8, g-lysozyme, viperin and invariant chain. The IL-8 cDNA full sequence of mandarin fish has been cloned using RACE-PCR and PCR. Its open reading frame has 297 nucleotides which encode a 99 amino acid peptide. The genomic sequence of interleukin-8 has been obtained by genomic walking. Its full sequence has 1643 nucleotides containing 4 exons and 3 introns. The deduced amino acid sequence of mandarin fish IL-8 was compared with those from other fish species. Four cysteines of CXC chemokines were identified at conserved locations. However, the mandarin fish IL-8 gene lacked the typical Glu-Leu-Arg (ELR) motif in the upstream of the fist pair of cysteines. The expression primers were designed to code for the open reading frames of IL-1β, IL-8, g-lysozyme, viperin and invariant chain and incorporate additional new restriction sites, respectively. The DNA products and vector pET-32a were digested by the two same enmyzes, and then ligated. After the recombinant plasmids were identified by sequencing, they were transformed into the prokaryotic cell Rosetta-gami(DE3). These recombinants were expressed by the chemical inducer IPTG. After the ultrasonic disruption, the expression products were analyzed by sodium dodecyl sulfate-polyacrylamide gel elactrophoresis (SDS-PAGE). The results indicated that IL-1?, IL-8, g-lysozyme, viperin and invariant chain were expressed efficiently in Rosetta-gami(DE3). The expression products were 43 ku, 25 ku, 35 ku, 55 ku, 45 ku fusion protein, respectively. G-lysozyme fusion protein was expressed in the natured form and purified by Ni-NTA affinity chromatography. Its purification showed the lytic activity against Micrococcus lysodeikticus. The other four fusion proteins, IL-1β, IL-8, viperin and invariant chain were expressed in the form of inclusion. They were purified in the denatured condition by utilizing affinity chromatography. Rabbit polyclonal antisera were developed against the purified IL-1β, IL-8, viperin and invariant chain recombinant proteins. Using Western blotting analyses, these proteins were recognized by the rabbit antisera, respectively. From the results of experiments, it has been deduced that the mandarin fish g-lysozyme expressed in E.coli has a value in application, and it provides independent evidence to analyze the potential role of the g-lysozyme in the innate immunity of fish. The rabbit antisera of IL-1?, IL-8, viperin and invariant chain fusion proteins have basis on analyzing the distribution of these genes in the mandarin fish tissues in vitro.