Cloning And Prokaryotic Expression Of IL-10 From Silver Carp, Hypophthalmichthys Molitrix

Interleukin-10 (IL-10) is an important anti-inflammation cytokine, functioning mainly in inflammation. In this study, IL-10 cDNA was cloned from silver carp (Hypophthalmichthys molitrix) using RACE (rapid amplification of cDNA ends)-PCR. The full IL-10 cDNA consisted of 1248 bp, including a 156 bp 5' untranslated region (UTR), a 552 bp 3'-UTR, and a 540 bp open reading frame (OPR) encoding a 179 amino acid peptide with four conservative cysteine (Cys) forming two pairs of disulphide bridges. As revealed by the reverse transcriptase PCR (RT-PCR), the IL-10 mRNA was mainly expressed in the spleen, gill, head kidney and muscle of the silver carp.The expression primers coding for the open reading frames of IL-10 were designed. The DNA products and vector pET-32a were digested by the two same enzymes, and then ligated to construct a prokaryotic expression vector pET-32a-IL-10 with a T7 promoter. The recombinant plasmid was examined by sequencing, and then transformed into the prokaryotic cell E. coli Rosetta-gami (DE3) for expression under the induction of IPTG After the ultrasonic disruption, the expression products were analyzed by sodium dodecyl sulfate-polyacrylamide gel elactrophoresis (SDS-PAGE). The IL-10 was expressed efficiently in E. coli Rosetta-gami (DE3) and the expression products were 39 kD fusion protein in the form of inclusion. The expressed protein was purified using HisBind Resin in the denatured condition and the purified protein was injected into rabbit in order to obtain polyclonal antisera. Using Western blot analyses, the recombinant protein was recognized by the rabbit antisera effectively. The results establish the foundation for further study of the function of IL-10.