基因重组BDNF蛋白在大肠杆菌中的表达纯化与功能鉴定

Expression,purification of recombinant BDNF protein in E.coli. and its functional characterization

目的构建脑源性神经营养因子(BDNF)基因的原核表达载体,并在大肠杆菌中进行表达,以获取高产量、低成本、高纯度且具有生物学活性的BDNF蛋白。方法以人的全长BDNFcDNA为模板,用PCR方法扩增成熟区BDNF的cDNA,应用基因重组技术将人BDNFcDNA克隆到质粒pET-30a(+)中,进行限制性内切酶酶切分析和DNA测序鉴定。将重组质粒转化大肠杆菌BL21(DE3)LysS,经IPTG诱导表达后,用Ni-NTA亲和层析纯化获取蛋白,用SDS-PAGE和westernblot方法鉴别,噻唑蓝(MTT)法检测重组蛋白对PC12细胞增殖的影响。结果扩增出的人BDNFcDNA片段克隆进了原核表达载体,经酶切和核酸测序鉴定,得到了正确的重组质粒pET-BDNF,并在大肠杆菌中获得了表达。纯化后的蛋白经考马氏亮蓝染色呈单一条带;用抗BDNF的抗体进行westernblot分析证明目的蛋白获得了表达。基因重组BDNF蛋白能够促进PC12细胞增殖。结论本研究成功构建了表达基因重组人BDNF的原核表达载体,基因重组人BDNF蛋白在大肠杆菌中获得了表达和纯化,所获得的基因重组蛋白具有较好的生物学活性。

Objective To construct the prokaryotic expression vector for brain-derived neurotrophic factor （BDNF） gene and express it in E.coli, so as to obtain the recombinant BDNF protein with high yield, low cost, high purity and high biological activity. Methods The cDNA fragment from maturation region of BDNF was obtained by standard PCR method with a full-length human BDNF cDNA as template, and was inserted into plasmid pET-30a（＋） by means of gene re-arrangement. The recombinant plasmid pET-BDNF was identified by restriction endonuclease analysis and DNA sequencing. The cloned pET-BDNF plasmid was transformed into E.coli host strain, BL21 （DE3）-LysS. Following induction by IPTG, the recombinant protein was expressed and then purified by Ni-NAT affinity chromatography under denaturing conditions. The interest protein was viewed by SDS-PAGE, further characterized by western blot. The proliferation of PC12 cells was evaluated by MTT assay. Results The human BDNF cDNA was amplified and cloned into prokaryotic expression vector. The restriction endonuclease analysis and DNA sequencing proved that the plasmid pET-BDNF was obtained. The recombinant protein was expressed in E. coli system. After Coomassie brilliant blue staining, the recombinant protein showed an exclusive band; western blot analysis with anti-BDNF antibody and anti-his antibody supported that the expressed protein was recombinant BDNF. The recombinant human BDNF protein could enhance the proliferation of PC12 cells. Conclusion The prokaryotic expression vector for human BDNF is successfully constructed. The recombinant human BDNF protein can be expressed and purified in E.coli. and has a good biological activity.