大鼠脑源性神经营养因子的克隆及原核重组表达载体的构建

目的:构建重组表达载体pGEX-脑源性神经营养因子(BDNF),探讨其原核表达BDNF的可行性。方法:大鼠BDNF进行密码子优化,人工合成BDNF的全基因序列,经回收、纯化、酶切后连接到pGEX原核表达载体中,将重组质粒pGEX-BDNF转化大肠杆菌BL21细胞中,筛选阳性克隆,经IPTG诱导表达后,利用ELISA方法鉴定其表达的活性。结果:PCR鉴定及测序显示BDNF序列完全正确,BDNF基因的克隆和表达载体构建成功,IPTG诱导表达目的蛋白经ELISA检测,其表达的目的蛋白具有一定的活性。结论:经密码子优化后经人工合成得到BDNF基因片段并成功构建pGEX-BDNF重组表达载体,具有一定的生物活性。

Clone of Rat BDNF Gene and Construction of Prokaryotic Recombinant Expression Vector

Objective： To construct and identify the prokaryotic recombinant expression vector pGEX-BDNF. Methods： The BDNF sequence from NCBI was optimized. The BDNF sequence was synthesized and ligated into pGEX prokaryotic expression vectors a~er recovery, purification and enzyme digestion. The purified recombinant plasmid was transformed into the E. coli BL21 cells. The positive clones were selected by PCR .The protein activity was identified by ELISA after IPTG induction. Results： PCR results and sequencing analysis showed that BDNF gene was correctly cloned into the prokaryotic recombinant expression vector pGEX. The ELISA results showed that the expression protein has a certain activity after IPTG induction. Conclusion： The recombinant prokaryotic expression vector pGEX-BDNF was successfully constructed.