中国人IL－9cDNA的亚克隆及其高效表达

Subcloning and High Level Expression of Chinese Human Interleukin-9cDNA

在完成了ｈＩＬ－９ｃＤＮＡ的克隆及序列测定的基础上，为实现ｈＩＬ－９ｃＤＮＡ在原核细胞中的表达，重新合成了上游引物（在酶切位，久后加上ＡＴＧ）。以ｐｕｃ１９－ｈＩＬ－９ｃＤＮＡ为模板进行了ＰＣＲ扩增，特异产物经ＥｃｏＲＩ酶切、回收后与经ｓｍａｌＩ、ＥｃｏＲＩ酶切的ｐＢｖ２２０载体连接，转化大肠杆菌。转化子质粒用０．８％ａｇａｒｏｓｅ电泳粗筛，经ＥｃｏＲＩ、Ｐｓｔｌ酶切后得到了３个阳性克隆。３个阳性克隆经热诱导后，在ＰａＰ＿Ｌ启动子作用下，获得了天然ｈＩＬ－９ｃＤＮＡ的高效表达，表达量分别达到可溶性总蛋白的３７．３％、４３．８２％、５４．５２％。

After cloning and sequencing of human IL-9 cDNA from Chinese peripheral monocytes,we amplifiedthe fragment encoding its mature form with ATG initiation codon,and subcloned it into the EcoR L ,Sma Isites of pBV22 0 expression vector.The transformants were primarily screened by plasmid size,thenidentified with EcoR I Pst I digestion。3 candidates were induced by temperature shift , and expressednative hIL- 9 at high level,i.e. up to 37.3%, 43.8%,and 54.5%of total soluble proteinsrespectively.