hIL-13融合表达载体的构建及其在大肠杆菌中的表达

THE CONSTRUCTION OF hIL-13 FUSION EXPRESSION VECTOR AND THE EXPRESSION OF IL-13 PROTEIN IN E.coli

通过ＲＴＰＣＲ技术直接从激活的健康人外周血单个核细胞（ＨＰＢＭＣ）中扩增出约０３ｋｂ的ｈＩＬ１３基因片段，经ＤＮＡ重组技术，插入到融合蛋白表达载体ｐＧＥＸ４Ｔ２中，转化入感受态的大肠杆菌ＴＧ１中，成功地构建了ｈＩＬ１３的基因工程表达菌株ｈＩＬ１３ｐＧＥＸ４Ｔ２／ＴＧ１。经异丙基硫代半乳糖苷（ＩＰＴＧ）诱导后表达的ＧＳＴＩＬ１３融合蛋白经ＳＤＳＰＡＧＥ证明分子量约３６ｋＤ，表达量约占菌体蛋白总量的１０％～３０％。复性后ＩＬ１３融合蛋白的生物学活性可达８０～１２０Ｕ／ｍｌ。复性后的融合蛋白经凝血酶作用后，可被切割成２６ｋＤ的ＧＳＴ与１０ｋＤ的ｈＩＬ１３。

The genetic engineering expression bacterial hIL13pGEX4T2/TG1 was successfully constructed through the insertion of hIL13 fragment(0.3 kb) that was directly amplified from activated healthy human peripheral blood mononuclear cells(HPBMC) by RTPCR method,into the GST fusion expression vector pGEX4T2,and the transformation into competent E.coli TG1.The molecular weight of GSTIL13 induced by IPTG is about 36 kD identified by SDSPAGE,and its expression level accounted 10%～30% of total bacterial proteins.The biological activities of renatured IL13 fusion protein could reach 80～120 U/ml and the fusion proteins could be cut into 26 kD GST and 10 kD IL13 with the treatment of thrombin for several hours.