【摘要】 目的克隆神经连接蛋白-1(Neuroligin-1,NL-1)细胞外基因片段1-691aa(NL1/1-691),插入载体pGEx-6p-1,并进行表达、纯化与鉴定。方法优化并常规合成基因NL1/1-691,添加5’BamHI和3’XhoI酶切位点,将基因通过5’BamHI and 3’XhoI酶切位点克隆至载体pGEX-6p-1(氨苄青霉素抗性)上,构建质粒pGEX-6p-1-NL1/1-691,筛选阳性克隆,转化至大肠杆菌C43感受态细胞中,蛋白质纯化筛选最佳诱导表达条件并获得目的蛋白;通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹(Western blot)对蛋白进行鉴定。结果经质粒双酶切鉴定、菌液PCR鉴定后,pGEX-6p-1-NL1/1-691原核表达载体构建成功。各个温度诱导下NL1/1-691蛋白均呈包涵体表达,表达优化后可将NL1/1-691从GST磁珠上纯化下来,得到部分可溶性蛋白,Western blot鉴定纯化蛋白为带GST标签的目的蛋白NL1/1-691。结论本实验室成功得到NL1/1-691重组蛋白,NL1/1-691最佳纯化条件为:37℃培养3 h加IPTG诱导为最佳诱导时机,IPTG诱导浓度为0.5 mmol/L,最佳诱导时间为12 h。将其应用到后续实验,有望为阿尔兹海默病的治疗提供新的思路。更多还原

【Abstract】 Objective To clone Neuroligin-1(NL-1) extracellular gene fragment 1-691 aa(NL1/1-691), and insert it into the vector pGEx-6 p-1, then carry out expression, purification and identification.Methods Optimize and synthesize the gene NL1/1-691 routinely, add 5’BamHI and 3’XhoI restriction sites, and clone the gene into the vector pGEX-6 p-1(ampicillin resistance) through the 5’BamHI and 3’XhoI restriction sites(Sexuality), construct plasmid pGEX-6 p-1-NL1/1-691, screen positive clones, transform into E. coli C43 competent cells, protein purification screen the best induction expression conditions and get the target protein, then use dodecyl sulfate Sodium-polyacrylamide gel electrophoresis(SDS-PAGE), Western blot(Western blot) was used to identify proteins.Results After the plasmid double enzyme digestion and the bacterial liquid PCR identification, the pGEX-6 p-1-NL1/1-691 prokaryotic expression vector was successfully constructed. NL1/1-691 protein was expressed as inclusion bodies under induction of various temperatures. NL1/1-691 could be purified from GST magnetic beads after expression optimization. Western results shown that the GST tag prokaryotic expression vector was successfully constructed. Conclusion Our laboratory has successfully obtained NL1/1-691 recombinant protein. The best purification conditions for NL1/1-691 are: culture at 37°C for 3 hours plus IPTG induction as the best induction time, IPTG induction concentration 0.5 mmol/L, and optimal induction time is 12 h. Applying it to follow-up experiments is expected to provide a new theoretical basis to treat Alzheimer’s disease.更多还原