融合蛋白pIL6-IL2在E.coli中的表达及活性鉴定

Expression of Fusion Protein pIL6-IL2 in E.coli and Bio-activity Assay

白细胞介素 2 (interleukin 2 ,IL 2 )与白细胞介素 6(interleukin 6,IL 6)能分别刺激T淋巴细胞增殖与B淋巴细胞分泌免疫球蛋白 ,从而促进动物机体的细胞免疫与体液免疫 ;另外IL2与IL6在发挥生物学活性时还有相互协同作用。因此 ,将去除信号肽的猪白细胞介素 6(pIL 6)与猪白细胞介素 2 (pIL 2 )cDNAs序列通过一段Linker相连 ,克隆到E .coli表达载体pPET 2 8a中。该融合蛋白IL6 IL2在E .coli表达菌BL2 1 (DE3)中获得成功表达 ,SDS PAGE分析分子量约为40kDa ,表达量达到总菌体的 66.2 6%。用IL6依赖的B9细胞与IL2依赖的CTLL细胞增殖试验进行融合蛋白IL6 IL2的生物活性检测 ,其活性可分别达到 0 8× 1 0 3 U mg和 6 4× 1 0 3 U mg。

Interleukin-2 and Interleukin-6 can stimulate T-lymphocytes proliferation and B-lymphocytes secreting immunoglobulins respectively,further enhance animal's cellular and humoral immune responses.Moreover,IL2 and IL6 can co-adjust biological activities between them.Hence,two cDNAs of the mature porcine IL6 (pIL6) and that of pIL2 were linked by a sequence with translating GGGGSEFGSGG and cloned into E.coli expression plasmid pET-28a.The fusion protein pIL6-IL2 was expressed in BL21 (DE3) with 40kDa of molecular weight by SDS-PAGE.The expression level reached to 66.26% and enhanced to 85.66% by purification.Analysis by cell proliferations of IL6-dependent cell B9 and IL2-dependent cell CTLL,the bio-activity units of the fusion protein IL6-IL2 can reach 0.8×103 U/mg and 6.4×103 U/mg respectively.;