弱化二氢叶酸还原酶基因增进人神经生长因子β亚基在CHO细胞中的表达

目的通过弱化二氢叶酸还原酶基因(Dihydrofolate reductase,DHFR)增进人神经生长因子β亚基(β-Humannerve growth factor,β-hNGF)在CHO细胞中的表达。方法从质粒pUC18-β-NGF中扩增β-NGF基因,克隆至pMD18-T载体,构建克隆质粒β-hNGF/pMD18-T;经人工合成弱化SV40启动子与DHFR基因顺式表达元件,并插入pBudCE4.1质粒,构建质粒w-DHFR/pBudCE4.1;分别双酶切质粒β-hNGF/pMD18-T和w-DHFR/pBudCE4.1后连接,转化大肠杆菌Top10’,构建重组表达质粒β-hNGF/w-DHFR/pBudCE4.1,转染CHO-DHFR-后,筛选阳性细胞克隆,ELISA法检测阳性CHO细胞株表达β-NGF的水平,鸡背根神经节增殖法检测重组β-NGF的生物学活性。结果重组表达质粒β-hNGF/w-DHFR/pBudCE4.1经酶切和测序证明构建正确;共获得5株表达β-hNGF的CHO细胞株,且表达的β-NGF能使神经节出现明显的神经纤维,表达量为0.1μg/μl。结论通过弱化SV40启动子和DHFR基因,在CHO细胞中成功表达了β-hNGF基因,表达量增加且具有生物学活性。

Attenuated dihydrofolate reductase gene enhances expression of β subunit of human nerve growth factor in CHO cells

Objective To enhance the expression of β subunit of human nerve growth factor(β-NGF)in CHO cells by attenuated dihydrofolate reductase(DHFR)gene.Methods β-NGF gene was amplified from plasmid pUC18-β-NGF,and cloned into vector pMD18-T to construct cloning vector β-hNGF / pMD18-T.The cis expression element for DHFR gene,with attenuated SV40 promoter,was synthesized artificially and inserted into plasmid pBudCE4.1 to construct recombinant plasmid w-DHFR / pBudCE4.1.Recombinant plasmids β-hNGF / pMD18-T and w-DHFR / pBudCE4.1 were digested with BstBⅠ and NheⅠ,than linked and transformed to E.coli Top10'.The constructed recombinant plasmid β-hNGF / w-DHFR / pBudCE4.1 was transfected to CHO-DHFRcells,and positive clones were screened and determined for expression level of β-NGF by ELISA,and for biological activity by chick embryo dorsal root ganglion proliferation test.Results Restriction analysis and sequencing proved that recombinant plasmid β-hNGF / pMD18-T and w-DHFR / pBudCE4.1 was constructed correctly.A total of five CHO cell strains expressing β-hNGF were obtained,and the expression level of β-hNGF was 0.1 μg / μl.Obvious nerve fiber appeared in chick embryo dorsal root ganglion treated with the expressed β-hNGF.Conclusion β-hNGF with biological activity was co-expressed in CHO cells by attenuation of DHFR gene,of which the expression level increased.