The Expression Of The ChIL-2 With And Without Signal Peptide In P.pastoris And Prokaryotic Expression System

Interleukin-2(IL-2) is a glucoprotein made by T lymphocyte cell and NK cell,which with important functions in the maturation,activation,proliferation and immune regulation of the immune cells.The ChIL-2 mRNA sequence with 747bp of length,429bp of reading frame of cDNA,143 of amino acid residues and 22 of amino acid residues at N-signal peptide sequence. The prokaryotic and P.pastoris expression vector were constructed and expressed in E.coli and P.pastoris in order to research the expression of the whole gene and the gene without signal peptide of the chicken interleukin-2 (ChIL-2) in eukaryotic and prokaryotic expression system.Three primers were designed according to the ChIL-2 gene sequences in the GenBank and the whole 429bp gene and 363pb gene without signal sequence of ChIL-2 were amplified from the induced spleen lymph cells of Lushi chicken embryo by using RT-PCR,which were cloned into the prokaryotic expression vector pET28a and P.pastoris express vector pPICZαA respectively to construct the prokaryon recombinate plasmid pET28a-IL-2,pET28a-△SIL-2 and P.pastoris recombinate plasmid pPICZαA-IL-2,pPICZαA-△SIL-2. Analyse by SDS-PAGE and Western blotting.Secondary structure was analyzed by RNA structure software.E.coli BL21(DE3) transformed with pET28a-IL-2 and pET28a-△SIL-2 had the highest expression level under the condition of 1.0mmol/L of IPTG for 4 hours, the protein with 22 kD and 18 kD were ChIL-2 and△S IL-2 respectively,the expression level of IL-2 is lower than△S IL-2 obviously at the same condition.ChIL-2 full-length gene formed a complex secondary structure at the 5′mRNA and the proportion of stem and ring was 1.034,while 0.571 of proportion of stem and ring with△SIL-2 5′mRNA.Maybe the complex and relatively stable 5′mRNA secondary structure of ChIL-2 obstruct the combination of mRNA with 30S subunit ribosome, and then influenced the starting frequency of translation.P.pastoris X-33 transformed with pPICZαA-IL-2 and pPICZαA-△SIL-2 had an obvious expression under the condition of 30℃and 1% of methanol after inducing 72h and 96h.The fusional protein with 28kD and 24kD were ChIL-2 and Ch△SIL-2,the expression level of IL-2 is a little lower than△SIL-2 at the same conditions ,and the former also formed a complex and relatively stable 5′mRNA secondary structure analyzed by RNA structure software.The proportion of stem and ring of IL-2 5′mRNA was 1,but 0.4706 of proportion of the latter.The complex secondary structure maybe one of the reason which effect the yeast express level.