猪IL-6 cDNA的克隆及在大肠杆菌中的高效表达

cDNA Cloning of Porcine Interleukin 6 and Its High Expression in E.coli

运用 RT- PCR方法 ,从经刀豆球蛋白 A(Con A)诱导的猪外周血单核细胞 (PMBCs)总 RNA中扩增得到了猪 IL -6 (porcine IL- 6 ,p IL- 6 )的 c DNA,并将其克隆到 p GEM- T载体上。DNA序列测定表明 ,克隆得到的 p IL- 6 c DNA与国外报道的完全一致 ,包括终止密码子在内其编码区的长度为 6 39bp,编码 2 12个氨基酸残基的蛋白质。将 p IL - 6成熟肽段编码区亚克隆至 p ET- 2 8(a)中 ,构建成原核表达质粒 p ETPIL6。该质粒的 BL2 1(DE3) L ys S转化菌在 IPTG的诱导下可高效表达 p IL- 6 ,表达量占菌体总蛋白的 30 .6 0 %～ 38.35 %。

The cDNA of porcine interleukin 6 (pIL 6)was amplified by RT PCR from the total RNA extracted from the peripheral blood lymphocytes stimulated with Concavadin and cloned into the pGEM T vector.Sequencing analysis showed that the encoding region of pIL 6 cDNA is 639 bp including the stop coden.A prokaryotic expression plasmid of pIL 6,pETPIL 6,was obtained by subcloning the encoding region of the pIL 6 mature peptide into pET 28a(+).The pIL 6 was expressed in pETPIL 6 transformed BL21(DE3)LysS induced by IPTG with the yield accounting for 30.60% 38.35% of the total bacterial protein.