【摘要】 为了构建一个含蛋白质剪接元件的表达载体 ,将 3.38kb的 p Ex Sec 的多克隆位点 (Eco R /Bam H )处插入一个含多个酶切位点的 49bp寡聚核苷酸。然后将 p MYB12 9中 intein- CBD基因片段移插入上述经改造的p Ex Sec I中 ,构建成含内蛋白子的表达载体 p Ex IC。根据人神经营养因子 - 3(h NT- 3)的已知 DNA序列 ,设计含 NdeI/Xho I酶切位点的引物 ,用 PCR法从人全血总 DNA中扩增 h NT- 3,再插入 p Ex IC载体中 ,构建成 p Ex IC- h NT- 3重组质粒。经转化后 ,工程菌 E.coli BL 2 1(DE3) /p Ex IC- h NT- 3在 L B培养基中获得融合表达 ,根据 intein的自剪切原理 ,在还原剂 DTT作用下 ,初步获得了约 14k D的 h NT- 3目的蛋白。更多还原

【Abstract】 In order to construct a fusion expression vector which contains the protein splicing element intein,a modified vector,pExSec I/Linker,was made from inserting a 49bp polymuleotides with several endonuclear cleavage sites into the site of EcoR I/BamH I in MCS of pExSec I.Then the new expression vector,pExIC was obtained with inserting the intein CBD fragment from pMYB129 into pExSec I/Linker.Meanwhile the recombinant plasmid,pExIC hNT 3 was constructed when hNT 3 gene which was amplified with human blood DNA as the template by PCR was inserted which was amplified with human blood DNA as the template by PCR was inserted into pExIC.After transformation,the strain E.coli BL21/pExIC hNT 3 was cultured in LB media and the fusion protein,intein hNT 3 was expressed after IPTG induction.According to the splicing principle of intein,the target protein,hNT 3 was occuried with DTT excision on SDS PAGE.更多还原