二连体人干细胞因子基因的构建及其在昆虫细胞中的表达

Expression of Covalently Linked Dimeric Human Stem Cell Factor in Sf9 Cells

应用DNA重组技术构建两端为人干细胞因子 (hSCF) 1～ 16 5肽编码区 ,中间为柔性连接肽 (GGGGSGGGGSGG)编码区的头尾相连的二连体基因 (dSCF) ,并将其插入杆状病毒转移载体 pVL1392中 .重组转移载体 pVL1392 dSCF与野生型苜蓿夜蛾核型多角体病毒AcNPVDNA共转染草地贪夜蛾细胞Sf9,经胞内同源重组和 4轮筛选获得纯净的重组病毒AcNPV dSCF .重组病毒感染Sf9单层培养细胞时 ,主要以分泌形式表达二连体蛋白 ,经MTT比色法测得dSCF的表达量约为 1180units/3× 10 6cells .WesternBlotting鉴定了昆虫细胞表达的重组人二连体干细胞因子 (rhdSCF) ,两种主要产物的分子量分别为 4 0kDa、4 9kDa .

Stem cell factor(SCF) is an early-acting hematopoietic cytokine that elicits multiple biological effects. It initiates signal transduction by direct dimerization of its receptor, c-kit, and the two juxtaposed receptors undergo tyrosine autophosphorylation, which leads to downstream intracellular signaling. SCF exists naturally as membrane-bound and soluble isoforms. The soluble SCF can form a non-covalently associated dimer, but under physiological conditions, greater than 90% of the circulating SCF exists as a monomer. E.coli-derived recombinant soluble hSCF variant, SCF(A165C), forms disulfide-linked dimer, which exhibits a 10-fold increase in the ability compared with the wild type monomer. The results suggest a model in which SCF dimerization mediates dimerization of its receptor, c-Kit, and subsequent signal transduction. When studying the binding of the dimeric hSCF to the extracellular domain of its receptor, c-Kit, Lemmom M A, et al, found that one SCF dimer bound simultaneously to two molecules of the c-Kit extracellular domain, which resulted in c-Kit dimerization, and provided additional supports for the latter model. In this essay, a covalently linked dimeric human stem cell factor(dSCF) was constructed which contained two human stem cell factors linked in a head_to_tail fashion by a short peptide (GGGGSGGGGSGG) by DNA recombinant techniques. The dSCF gene was cloned into the baculovirus transfer vector pVL1392, yielding a plasmid pVL1392-dSCF. Then the plasmid was cotransfected with wild-type Ac- NPV DNA into Sf9 cells by a lipofectin-mediated procedure. After four cycles of dot hybridization, the recombinant viruses were isolated. The one with the highest activity was chosen for further study and designated AcNPV-dSCF. The recombinant protein was expressed in Sf9 cells infected with AcNPV-dSCF and mainly secreted into the culture medium, with the highest activity of about 1?180?units?/?3×10 6?cells on the second day (after infection). Two main bands with molecular masses of 40?kDa and 49?kDa were detected by Western Blotting.