论文部分内容阅读
通过ＲＴＰＣＲ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０３ｋｂ的ｈＩＬ１３基因片段，经ＤＮＡ重组技术，插入到融合蛋白表达载体ｐＧＥＸ４Ｔ２中，转化入感受态的大肠杆菌ＴＧ１中，成功地构建了ｈＩＬ１３的基因工程表达菌株ｈＩＬ１３ｐＧＥＸ４Ｔ２／ＴＧ１。经异丙基硫代半乳糖苷（ＩＰＴＧ）诱导后表达的ＧＳＴＩＬ１３融合蛋白经ＳＤＳＰＡＧＥ证明分子量约３６ｋＤ，表达量约占菌体蛋白总量的１０％～３０％。复性后ＩＬ１３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ｈＩＬ１３。
By RT PCR technology directly from activated human peripheral blood mononuclear cells (HPBMC) amplified hIL 13 gene fragment of about 0 3kb, recombinant DNA technology was inserted into the fusion protein expression vector pGEX 4T 2 Transformed into competent E. coli TG1 successfully constructed hIL 13 genetically engineered strains hIL 13 pGEX 4T 2 / TG1. The GST-IL-13 fusion protein induced by isopropylthiogalactopyranoside (IPTG) showed a molecular weight of about 36 kD by SDS-PAGE, and the expression amount was about 10% -30% of the total bacterial protein. Refolding IL 13 fusion protein biological activity of up to 80 ~ 120U / ml. Refolded fusion protein after thrombin, can be cut into 26kD GST and 10kD hIL 13.