【摘要】 目的　为进一步研究神经生长因子 (β- NGF)及其基因的结构及功能奠定基础。方法　自行设计合成一对特异引物 ,直接从人血细胞基因组 DNA中 PCR扩增出 NGF基因片段 ,克隆至 p GEM- T Easy载体 ,p GEM- T- NGF克隆片段再定向插入原核表达载体 p GEX- 5 T,构建的重组体 p5 TNF转化大肠杆菌 JM10 9并经IPTG诱导表达。结果　经限制性酶谱和 DNA测序确证 ,p GEM- T- NGF克隆片段为 β- NGF全长编码序列(380 bp) ;SDS- PAGE、分光光度计扫描和 Western印迹显示 ,p5 TNF转化菌有 40 .5× 10 3u特异区带 ,融合蛋白表达量为 5 0 3.2 mg/ L,占菌体可溶性蛋白总量 (7.4g/ L) 6 .8% ,该产物具 NGF抗原活性。结论　本研究成功克隆 β-NGF基因全长编码序列并在大肠杆菌中获稳定表达 ,同时证实国人 β- NGF基因编码无特殊性更多还原

【Abstract】 Objective To investigate the structure and function of human nerve growth factor(β NGF) and the gene encoding β NGF. Methods A pair of specific primers (29mer) for the sequence encoding human β NGF was designed and synthesized. A 380bp fragment was amplified from human blood genomic DNA by polymerase chain reaction, and cloned into pGEM T Easy vector. The identified insert fragment from the recombinant pGEM T NGF was directionally ligated with linearized pGEX 5T with the compatible termini. E.coli JM109 was transformed with the expression recombinant p5TNGF and induced by IPTG. Results The cloned DNA fragment was identified as the full length sequence encoding human β NGF by restriction analysis and DNA sequencing. SDS PAGE and Western blot revealed the cloned NGF gene expressed as a fusion protein (40.5×10 3 u) in the cells transformed by p5TNGF. The soluble fusion protein was determined to be 503.2mg/L, accounting for 6.8% of the total soluble protein (7.4g/L) of bacterial cells. This fusion protein was found to have antigenic activities of NGF. Conclusion The clone containing the full length sequence encoding human β NGF is obtained and successfully expressed in E.coli to be of use for studying the biological functions of human β NGF gene.更多还原