Gene Clonging, Expression And Biological Activity Of Porcine Interleukin18

In this study,the porcine interleukin 18 gene has been investigated, then subcloned it into prokaryote and eukaryotic expression vectors,and the research on interleukin18 biological activities has paved the way for practical use of interleukin 18.According to interleukin 18 published in GenBank,pair of primers were designed. Using the primers,full-length interleukin 18 was amplified by RT-PCR.It was cloned into pGEM-T vector and then sequenced.The result showed that the full-length interleukin 18 composed 579 bp,which encodes 192 amino acids with a 35-amino-acid signal peptide.And the sequence No.in GenBank is DQ 499825。According to interleukin 18 nucleotide sequence,pairs of primers were designed. The mature protein gene was amplified by PCR.It was cloned into the prokaryote expression vectors pGEX-6P-1,pET-28a and pQE30,then obtained recombinant plasmids pGEX-IL18,pET-IL18,pQE-IL18 respectively,pGEX-IL18 and pET-IL18 could expressed in E.coli BL21 and pQE-IL18 could expressed in E.coli JM109 via IPTG induction.SDS-PAGE showed that pGEX-IL18 fused protein was about 45 kDa in size,and it accounted for 28%of the total bacterial protein when it expressed highest,pET-IL18 fused protein was about 22 kDa in size,and it accounted for 27% of the total bacterial protein when it expressed highest.pQE-IL18 fused protein was about 19 kDa in size,and it accounted for 17%of the total bacterial protein when it expressed highest.After being degenerated and then renaturated,the activity of the pGEX-IL18 inclusion body was detected by MTT and inhibiting the cytopathic effect.As a result, IL-18 can improve the lymphocytes activity and the activities of recombinant IL-18 against PRV,PPV at PK15 cell are 2.00×10~3U/mg and 2.24×10~3U/mg respectively, the activity of recombinant IL-18 against PRRSV at Marc145 cell is 2.50×10~3U/mg.The whole interleukin 18 fragment was subcloned into the eukaryotic expression vector pcDNA3.1 to construct recombinant plasmid pcDNA-IL18.And then the recombinant plasmid was transfered on the PK15 cells by lipofectamine 2000. RT-PCR identification indicated that the fragment had already inserted into PK15 cell chromosome,pcDNA-IL18,pcDNA3.1,PRV live vaccine,pcDNA-IL18 added PRV live vaccine were inoculated instramuscular respectively in small white mice two times at fourteen days intervals.While it had a control group.Peripheral blood lymphocytes of immunized group were detected by MTT at one week interals from one to eight week post-vaccination.The result showed that the peripheral blood lymphocytes activities of pcDNA-IL18 and pcDNA-IL18 added PRV live vaccine were higher than other groups.To summarize,interleukin 18 gene was obtained,prokaryote expression plasmids of pGEX-IL18,pET-IL18,pQE-IL18 and eukaryotic expression plasmid of pcDNA-IL18 were constructed successfully,prokaryote expression proteins were obtained.The result of the research demonstrated that interleukin 18 can enhance the cellular immune responses and antivirus.