人BDNF在CHO及大肠杆菌中的表达及功能研究

发布时间：2018-07-18 12:04

【摘要】： 目的:将人脑源性神经生长因子(Brain-derived neurotrophic factor, BDNF)基因在真核及原核表达系统中进行重组表达,并检测所表达蛋白的功能,为开发高活性的hBDNF基因重组蛋白和基因治疗载体奠定基础。方法:将由本科室构建和保存的人BDNF基因真核表达载体pTracerTM- EV/V5-His-hBDNF鉴定后导入CHO细胞中进行表达;同时将本科室所保存的质粒pBV220-BDNF的BDNF基因酶切后重组到表达效果更高的原核载体pET3.0a+上,鉴定后导入原核细胞中进行表达。并用SDS-PAGE、RT-PCR、Western-blot、MTT法检测促PC12细胞生长等方法进行其抗原性和生物学活性的检测。结果:原核载体重组构建成功。原核和真核表达载体分别在大肠杆菌和CHO细胞中均获成功表达。在大肠杆菌中表达的包涵体蛋白约占菌体总蛋白的37.9%,通过透析复性后,获得具有活性的表达蛋白。该复性后蛋白和CHO细胞表达产物均具有较好的抗原活性促进PC12细胞生长的生物学功能。结论:人BDNF基因在CHO细胞和大肠杆菌中均获成功表达,表达蛋白具有良好的抗原性和生物学活性,此实验为生物工程技术研制rhBDNF蛋白和基因治疗载体提供了依据。
[Abstract]:Aim: to investigate the expression of brain-derived neurotrophic factor (BDNF) gene in eukaryotic and prokaryotic expression systems, and to detect the function of the expressed protein in order to lay a foundation for the development of highly active recombinant hBDNF gene protein and gene therapy vector. Methods: the eukaryotic expression vector pTracerTMEV-V5-His-hBDNF constructed and preserved by our department was transfected into Cho cells for expression, and the BDNF gene of plasmid pBV220-BDNF was digested into pET3.0a. After identification, it was introduced into prokaryotic cells for expression. The antigenicity and biological activity of PC12 cells were detected by SDS-PAGEG RT-PCRP-Western-blot MTT assay. Results: the prokaryotic vector was successfully constructed. Prokaryotic and eukaryotic expression vectors were successfully expressed in E. coli and Cho cells, respectively. The inclusion body protein expressed in Escherichia coli accounted for about 37.9% of the total bacterial protein. After dialysis renaturation, the active expressed protein was obtained. Both the refolding protein and the Cho cell expression products have good antigenic activity to promote the growth of PC12 cells. Conclusion: human BDNF gene was successfully expressed in Cho cells and Escherichia coli, and the expressed protein had good antigenicity and biological activity. This experiment provides a basis for the development of rhBDNF protein and gene therapy vector by bioengineering.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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