【摘要】： 目的:將人腦源性神經(jīng)生長因子(Brain-derived neurotrophic factor, BDNF)基因在真核及原核表達系統(tǒng)中進行重組表達,并檢測所表達蛋白的功能,為開發(fā)高活性的hBDNF基因重組蛋白和基因治療載體奠定基礎(chǔ)。 方法:將由本科室構(gòu)建和保存的人BDNF基因真核表達載體pTracerTM- EV/V5-His-hBDNF鑒定后導入CHO細胞中進行表達;同時將本科室所保存的質(zhì)粒pBV220-BDNF的BDNF基因酶切后重組到表達效果更高的原核載體pET3.0a+上,鑒定后導入原核細胞中進行表達。并用SDS-PAGE、RT-PCR、Western-blot、MTT法檢測促PC12細胞生長等方法進行其抗原性和生物學活性的檢測。 結(jié)果:原核載體重組構(gòu)建成功。原核和真核表達載體分別在大腸桿菌和CHO細胞中均獲成功表達。在大腸桿菌中表達的包涵體蛋白約占菌體總蛋白的37.9%,通過透析復性后,獲得具有活性的表達蛋白。該復性后蛋白和CHO細胞表達產(chǎn)物均具有較好的抗原活性促進PC12細胞生長的生物學功能。 結(jié)論:人BDNF基因在CHO細胞和大腸桿菌中均獲成功表達,表達蛋白具有良好的抗原性和生物學活性,此實驗為生物工程技術(shù)研制rhBDNF蛋白和基因治療載體提供了依據(jù)。
[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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R392

【引證文獻】

相關(guān)碩士學位論文 前1條

1 黎威;多價血吸蟲蛋白疫苗的優(yōu)選與原核表達研究[D];華中科技大學;2012年

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