摘要
目的克隆大鼠脑组织神经营养素3(NT-3)基因,原核诱导表达并纯化,制备高效价抗体,为研究其对周围神经损伤修复的影响提供实验基础。方法用RT-PCR法扩增目的基因,依次克隆入pMD-18T载体及亚克隆入pRSET-A原核表达载体。导入大肠杆菌BL21经IPTG诱导表达,SDS-PAGE电泳鉴定后用镍柱纯化。免疫家兔制备特异性抗体。结果RT-PCR得到777 bp的片断,酶切鉴定及测序证实与GeneBank中大鼠NT-3序列一致,成功构建了原核表达载体pRSET-A-NT-3。导入BL21诱导得到一条约34 ku的新蛋白带。免疫家兔获得1∶64000的高效价特异性抗大鼠NT-3血清。
Objective To clone NT-3 gene from normal rat brain and to purify its fusion protein and to prepare specific high liter antibody so that to provide a foundation for further study for peripheral nerve injury. Methods We amplified target gene by RT-PCR and cloned it into the vector of pMD-18T, then analyzed its sequence and compared it with the sequence from GenBank. We subcloned it into pRSET-A vector and introduced it into Escherichia coli BL21. The expression was induced by IPTG, and identified by SDS-PAGE. The fusion protein was purified by niecolum purify kit. We immuned rabbits with immunological adjuvant for specificity antibody preparation. Resuits We got a 777 bp gene segment by RT-PCR. The DNA sequence was identical to rat NT-3 gene sequence in GenBank. It proved that the target gene was correctly inserted into the vector. A new protein band of about 34 ku appeared on SDS-PAGE after induction of IPTG. A specific high titer antibody of 1 ： 64000 was gained by immunizing rabbits with adjuvant.
出处
《基础医学与临床》
CSCD
北大核心
2007年第3期324-328,共5页
Basic and Clinical Medicine
基金
国家自然科学基金(30271315)
