重庆医学2016，Vol.45Issue(20)：2737-2739,3.DOI:10.3969/j.issn.1671-8348.2016.20.001
脑源性神经营养因子原核表达载体的构建及其生物学活性分析
Research on construction of prokaryotic expression vector of brain-derived neurotrophic factor and its biologic activity
摘要
Abstract
Objective To construct the prokaryotic expression plasmid pET 28a(+ )/BDNF and corresponding prokaryotic expression bacterium containing brain‐derived neurotrophic factor(BDNF) ,and to research its biologic activity after IPTG induction expression .Methods The codon optimization was done according to Escherichia coli (E .coli) BL21(DE3) bias .The rat BDNF gen sequence was then artificially synthesized and the prokaryotic expression plasmid pET 28a(+ )was constructed .After identification by the restriction enzyme digestion (Bam HI ,Hind Ⅲ)and DNA sequencing ,E .coli BL21(DE3) was introduced .After IPTG induc‐tion expression ,the recombinant protein expression was detected by SDS‐PAGE ,the BDNF protein antigenicity was indentified by Western blot with anti‐BDNF specific antibody .MTT assay was used to evaluate the influence of BDNF protein on the PC 12 cell vi‐ability .Results The constructed plasmid of pET28a(+ )/BDNF was 100% consistent with the designing sequence confirmed by re‐striction enzyme digestion and DNA sequencing identification ;the engineering bacterium had a clear protein band at the relative mo‐lecular mass about 17 × 103 after IPTG induction ,Western blot displayed that the target protein could react with specific BDNF an‐tibody ;compared with the normal control group ,the cell viability in the different concentrations of BDNF protein treatment groups was increased to some extent compared with the control group ,in which 50 ng/mL BDNF treatment group showed the best effect . Conclusion The prokaryotic expressing plasmid pET28a(+ )/BDNF and corresponding prokaryotic expressing bacterium are con‐structed successfully and the BDNF protein expressed by this engineering bacterium has a good biologic activity .关键词
脑源性神经营养因子/原核表达/生物学活性Key words
brain-derived neurotrophic factor/prokaryotic expression/biologic activity分类
医药卫生引用本文复制引用
李健春,王丽,姜宁,王琼..脑源性神经营养因子原核表达载体的构建及其生物学活性分析[J].重庆医学,2016,45(20):2737-2739,3.基金项目
人因工程重点实验室开放基金资助项目（ H F2012-K-06）；四川省科技厅-泸州市人民政府-泸州医学院联合科研资金项目（14JC01323-LH50）；国家科技重大专项（2012ZX09J12201）。 （）
