Research On The Gene Synthesis, Prokaryotic Expression & Preliminary Biological Activity Of HIL-18

Interleukin-18(IL-18) was first purified by Okamura,H in 1995 from the livers of mice treated with the bacterium P.acnes and challenged with lipopolysaccharide(LPS) to induce toxic shock. Human interleukin-18(hIL-18),whith is a potential anti-tumor and antimicrobiol drug,prossesses potent biological activities,including the induction of IFN-γproduction in T cell,the promotion of T cell proliferation,and enhancement of NK cell cytotoxicity.hIL-18 mature peptide was containing 157 amino acid residues , the molecular weight of 18.3kDa, although the existence of 4 cysteine, but no disulfide bond formation, to play a biological role of the monomer form.The purpose of the research is to obtain high quality and quantity of recombinant hunan Interleukin-18(rhIL-18) in Escherichia coli expression system, to achieve this, the principal has taken the following optimization strategy:When the foreign gene contains more rare codons, will affect its high-level expression in Escherichia coli,the sequence of mature hIL-18 has 37aa rare condons for E.coli in a total of 157aa. To overcome this problem, Gene Designβ2.1 software to use to optimize the design of the hIL-18 gene, hIL -18 in the original gene replacement of rare codons become synonymous codon preferences.16 oligonucleotides were designed and synthesized,the whole DNA sequence was synthesized by two-step method(DA-PCR and OE-PCR), the synthesized fragments were cloned into EcoRⅠ/PstⅠsites of pUC18, transformed into the E.coli JM109 cells. Screening of positive clones for sequencing,and obtain the correct hIL-18 gene,gene synthesis error rate of 5.31‰.The hIL-18 DNA fragments were cloned into HindⅢ/BglⅡsites of pET32a(+), the hIL-18 was in a high level of expression in E.coli BL21 (DE3).In shake flask,46% expression ratio was achieved,according to the conseruence of SDS-PAGE analysis.Immunology activity was identified by western blot. The optimal conditions of the protein induction are as follows: when the absorbance of the E.coli BL21(DE3) at 650nm is 0.6,add IPTG to the final concentration 0.1mM. then culture the strain at 37℃for 4h and harvest the cells. The main pattern of the expressive target protein in the cell is soluble.from the calculation, the percentage of the target protein in the total protein exised in the supernatant is about 27%.We established a method to determine the biologic activity of hIL-18 mature peptide by using the spleen cell of the mice. And to verify that:the hIL-18 mature peptide digested by enterokinase can stimulate the spleen cell to produce IFN-γ,and it has biologic activity.