摘要
目的分离人胶质细胞源性神经营养因子(hGDNF)成熟肽基因,并在E.coil中获得表达。方法以人的白细胞基因组DNA为模板,合成特异引物,采用PCR技术分离得到hGDNF成熟肽基因,并构建表达载体pET-21a(+)-hGDNF,转化大肠杆菌BL21(DE3),分别用IPTG和乳糖诱导表达,SDS-PAGE电泳检测hGDNF蛋白表达。结果PCR扩增出了400bp的DNA片段,构建的表达载体pET-21a(+)-hGDNF经酶切、PCR鉴定均出现400bp左右的条带,测序结果正确,用IPTG或乳糖诱导转化菌均有15kd大小的目的条带,表达的目的蛋白占菌体总蛋白30%以上,以包涵体形式存在。结论克隆得到人GDNF成熟肽基因并在大肠杆菌中得到了高效表达。
Objective To isolate the gene encoding human glial cell line-derived neurotrophic factor (hGDNF) mature peptides and to express it in E. coli. Methods The gene of hGDNF mature pep-tides was cloned by PCR method from human genomic DNA with primers designed according to the data in GeneBanK. After proved by gene sequencing, the PCR fragment was subcloned into the E. coli expression vector pET-21a( + ), and transterred into E. coli BL21(DE3). The expression of hGDNF protein in transformant was controlled by T7 promotor with IPTG or lactose. Results hGDNF DNA fragment with 400 bp was obtained. Recombiant protein was expressed in E. coli host strain BL21 (DE3) in the form of inclusion body, and the expression level was more than 30% of the total cell lysate. The hGDNF amounted to at least 80% after purification. Conclusion The gene of hGDNF is successfully cloned and an E. coli strain high expressing hGDNF is obtained.
出处
《苏州大学学报（医学版）》
CAS
北大核心
2005年第1期34-37,共4页
Suzhou University Journal of Medical Science
