bFGF在原代培养人皮肤成纤维细胞中的高效分泌表达

High-level secretory expression of bFGF in primarily cultured human skin fibroblast

目的:提高碱性成纤维细胞生长因子在原代培养的成纤维细胞中的分泌表达。方法:bFGF缺乏典型的分泌信号肽,将鼠抗体轻链基因Igκ的信号肽序列引入该基因的5′端得到新的Igκ-bFGF重组体基因。同时考虑到人源细胞氨基酸编码密码子的偏好性,对整个基因序列密码子的第3位摇摆碱基作了偏好性调整,并克隆入真核表达载体中。得到的重组质粒用酶切的方法线性化,尽量去除质粒载体上大肠杆菌起源的基因序列,然后通过电穿孔的方法将线性质粒转导入原代培养的人皮肤成纤维细胞。结果:与野生型的bFGF基因相比,嵌合体bFGF被大量分泌到培养基中,同时对于成纤维细胞的增殖有明显的促进作用。结论:这种嵌合体基因的构建可以明显提高成纤维细胞对bFGF的分泌表达。

Aim: To improve the expression level of bFGF in primarily cultured human skin fibro(blast. )Methods: The bFGF lacks a classical secretion signal peptide, a novel recombinant Igκ-bFGF was constructed by placing the signal sequence of mouse antibody light chain Igκ upstream the bFGF gene. Considering the codon usage biases in human cells, the whole sequence of this chimera gene was optimized to improved the expression level. Then it was transducted into primarily cultured human skin fibroblast by electroporation, before which the recombinant was digested into linearized vector, in order to remove the E.coli-derived sequences. Results: The Igκ-bFGF gene was secreted into the media more highly compared with wild type bFGF, and was able to activate the growth of fibroblasts effectively. Conclusion:These results indicate that this novel Igκ-bFGF fusion gene may be markedly secreted from fibroblasts.