重组人G-CSF双体分子的构建表达及生物活性和结构特性分析  

Construction and expression, biological activities and protein characteristics analysis of human recombinant bimolecular G-CSF

作　　者：郭斯启[1] 奚永志[1] 赵丹丹[1] 孙玉英[1] 崔建武[1] 刘楠[1] 梁飞[1] 

机构地区：[1]军事医学科学院附属医院免疫学研究室及国家生物医学分析中心免疫分析实验室,北京100039

出　　处：《生物技术通讯》2003年第6期489-493,共5页Letters in Biotechnology

基　　金：国家自然科学基金资助(3980056)

摘　　要：研究目的是分子设计并构建较G-CSF单体分子具有半衰期更长、生物活性更高的新型重组人G-CSF/G-CSF双体分子(简称G-G),并在原核系统进行高效表达。分别构建pET32/G-G和pET32/G原核表达载体,实现G-G双体融合蛋白在大肠杆菌中的高效表达,利用亲和层析等方法进行蛋白纯化;对该融合蛋白的结构特征诸如等电点、柔性、抗原性及亲水性进行模拟分析;采用MTT法对G-G双体融合蛋白的生物学活性进行测定。结果首次构建成功了G-G双体分子融合蛋白高效表达载体,表达量高于40％,一步亲和层析所获融合蛋白的纯度为80％左右。该融合蛋白的结构特征模拟分析的结果显示,G-CSF双体分子的等电点、柔性、抗原性及亲水性均未显著改变。活性测定表明,所构建表达的重组人G-G双体分子能有效刺激G-CSF依赖细胞株NFS-60的增殖,但其刺激效应低于G-CSF的单体和标准品。结果表明,所构建表达的G-CSF的单体和G-G双体分子均可在大肠杆菌中获得高效表达,但G-G双体分子的比活性不及G-CSF单体分子,与预期设想的有差别,其原因正在研究之中。Molecule design, construction and high level expression in E. coli. of bimolecular G-CSF with longer half life and higher bioactivities than single molecular G-CSF. The expression vectors of G-CSF and bimolecular G-CSF (named G-G), pET32 a(+)/G-CSF and pET32 a( + )/G-G, were constructed. Then, they were expressed in host bacterium, and purified with affinity chromatography. Protein characteristics of G-G, such as second structure, antigenici-ty, isoelectric point, hydrophilicity and flexibility are predicted. The bioactivities of G-CSF and G-G were identified by MTT test in vitro with NFS-60 cells dependent on G-CSF. The expressing vectors pET32a(+)/ G-CSF and G-G were constituted correctly and expressed in origamiTM (DE3), and their expression efficiency were more than 40 percent of total protein. Protein characteristics prediction of G-G manifests its second structure, antigenicity, isoelectric point, hydrophilicity and flexibility are no significant change. The G-G can stimulate the proliferation of NFS60 cell line, but its bioactivity is less than that of single molecular form and the standard sample of G-CSF. Both G-CSF and G-G can express high level in E.coli, but the bioactivity of G-G in vitro is less than that of single molecular form of G-CSF.

关 键 词：融合构建 分子特性预测 大肠杆菌 高效表达 造血刺激活性 G—CSF双体分子 

分 类 号：Q786[生物学—分子生物学]