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利用PCR扩增得到粒细胞 巨噬细胞集落刺激因子 (GM CSF)、白细胞介素 3(IL 3)完整基因片段 ,将其分别克隆至pGEM T ,构建成GM CSF/IL 3融合蛋白基因 ,DNA序列与设计预期一致。将得到的融合蛋白基因克隆至T7RNA聚合酶表达载体pT7zz,得到表达质粒pFu ,经转化至表达宿主E .coliBL2 1(DE3) ,在IPTG诱导下获得融合蛋白目的产物的直接表达。经SDS PAGE电泳鉴定扫描分析 ,目的基因产物表达量占菌体总蛋白量的 30 %以上 ,目的基因表达产物以包涵体的形式表达。Western blot鉴定表明 ,该表达产物可以与GM CSF抗体及IL 3抗体特异性结合。目的基因表达产物经过包涵体变性、透析复性及柱层析纯化 ,用GM CSF、IL 3依赖细胞株TF 1检测 ,具有明显的生物学活性
The complete gene fragment of GM-CSF and IL-3 was amplified by PCR and cloned into pGEM T respectively to construct GM CSF / IL 3 fusion protein gene. The sequence is in line with the design expectation. The obtained fusion protein gene was cloned into the T7 RNA polymerase expression vector pT7zz to obtain the expression plasmid pFu which was transformed into the expression host E. coli BL21 (DE3) to obtain the direct expression of the target product of the fusion protein under the induction of IPTG. SDS PAGE electrophoresis identification scanning analysis, the target gene product expression amount accounted for more than 30% of the total bacterial protein, the target gene expression product was expressed in the form of inclusion bodies. Western blot analysis showed that the expressed product could specifically bind to GM CSF antibody and IL 3 antibody. The expressed product of the target gene was purified by inclusion body denaturation, dialysis refolding and column chromatography, and was detected by GM-CSF and IL-3-dependent cell line TF 1 and had obvious biological activity