人IL-6基因的克隆及其在原核生物中表达及条件的优化  

Cloning of Human Interleukin-6 Gene and the Optimized Expression Condition in Prokaryotes

作　　者：史继静[1] 刘朝奇[1] 杨凡[1] 杨祖伟[1] 

机构地区：[1]三峡大学分子生物学研究所,宜昌443002

出　　处：《生物技术通报》2010年第5期135-140,共6页Biotechnology Bulletin

摘　　要：构建人白细胞介素6(IL-6)的原核表达载体并优化其表达条件,为IL-6的高效表达提供试验依据。以人T细胞cDNA为模板通过PCR方法扩增IL-6基因,将其克隆到原核表达载体pET28a(+)中,酶切及测序鉴定重组体。将构建好的重组质粒转化大肠杆菌BL21(DE3),用IPTG进行诱导表达,产物用Western blotting及人IL-6检测试剂盒分析鉴定。在保持菌种不改变的前提下,分别改变IPTG的浓度、培养时间、卡那霉素浓度、培养温度等来优化IL-6表达条件。结果显示,原核表达载体pET28 a(+)-IL-6成功构建,可在大肠杆菌BL21(DE3)中诱导表达,得到相对分子质量约22 kD的IL-6蛋白,经Western blotting鉴定正确,经人IL-6试剂盒检测显示具有较高的免疫活性。在IPTG浓度400μmol/mL,卡那霉素浓度50μg/mL,40℃培养6 h的条件下,目的蛋白表达量最高,可占总蛋白表达量的40%。成功构建人IL-6原核表达载体且获高效表达,为研究IL-6生物学活性及产品开发提供了试验基础。The study was designed to optimize human interleukin-6（]L-6）expression in prokaryotes for the high effective expression. The full length gene fragment of hIL-6 was amplified by PCR,and then cloned into prokaryotic expressing vector pET28a（ ＋ ）. The recombinant protein IL-6 was induced by IPTG in E. coli BL21 （DE3） and the expressed protein was detected by Western blot as- say and ELISA. Through changing the concentration of IPTG and kanamycin,length of time induced by IPTG,and cultured temperature to optimize IL-6 expression. Results indicated that the plasmid pET28a（ ＋ ）-IL-6 was successfully constructed and hlL-6 protein could be expressed in E. coli BL21 （DE3）. The recombinant protein was characterized by western blotting and ELISA. Through optimizing, the satisfactory expression condition was obtained,as follow including IPTG concentration 400 μ mol/mL;induced time of 6 hours;kanamycin 50 μg/mL; cultured temperature 40℃. The plasmid pET28a（ ＋ ）-IL-6 was successfully constructed and expressed in pro- karyotes with high efficiency,which provided reliable tools for further studying IL-6 biological activities and exploitation as manufactures.

关 键 词：IL-6 原核表达 条件优化 

分 类 号：Q78[生物学—分子生物学]