影响人干细胞因子基因高效表达的因素探讨  

Study on high-level expression of human stem cell factor gene in E. coli

作　　者：洪海燕[1] 戚中田[1] 赖春宁[2] 冯健男[2] 王建安[2] 刘 涛[2] 沈倍奋[2] 

机构地区：[1]第二军医大学微生物学教研室,上海200433 [2]军事医学科学院基础医学研究所分子免疫室,北京100850

出　　处：《免疫学杂志》2002年第3期225-228,共4页Immunological Journal

摘　　要：目的 分析SD序列长度、基因起始码ATG和终止码TAA上下游处RNA的二级结构及密码子偏性等影响表达效率的主要因素,以期实现重组人干细胞因子在原核细菌中高效表达。方法 分析并修改编码人SCF氨基酸的密码子,分段合成改造后的基因,PCR一次性完成拼接,将基因克隆到pBV-220载体上,诱导表达。结果 天然人SCF基因在pBV-220载体的表达占菌体总蛋白的9.5％,基因改造后SCF占菌体总蛋白的27％,表达的蛋白能够为SCF的单克隆抗体所识别,初步纯化的SCF蛋白纯度大于80％,能够促进Mo7e细胞的增殖活性。结论 密码子偏性是影响SCF在原核细菌中高效表达的主要因素,通过改变密码子偏性能过实现高效表达。ve To highly express human stem cell factor (hSCF) in bacterial strain, three main factors( i.e. lengths of SD, secondary structure of the connection between gene and vector, amino acid codon bias ) that contribute to high expression were studied. It is the amino acid codon adaptive that make SCF low expression. Method The gene of hSCF was corrected and primer was designed with the help of Goldkey software. The gene was synthesized, ligated by PCR and cloned into a prokaryotic temperature sensitive vector pBV-220. Results Af-ter induction, the recombinant bacteria could produce rhSCF about 27 percent of all bacterial protein. Western-blot showed a stained band at 19 000 u. The biologic assay showed that rhSCF could stimulate the proliferation of Mo7e cells. Conclusion It is the amino acid codon bias that make SCF low expression.

关 键 词：pBV-220 RNA 二级结构 蛋白表达 全基因合成 干细胞因子 

分 类 号：Q786[生物学—分子生物学]