摘要
为了得到人源性神经营养素-6(N eurotroph in-6,NT-6)成熟肽片段,本研究以经测序鉴定的人源性NT-6 cDNA为模板,通过聚合酶链式反应(Po lym erase cha in reaction,PCR)扩增得到NT-6蛋白成熟肽编码区基因片段;将该基因克隆到融合表达载体pGEX 1-λT,构建重组原核融合表达质粒pGEX-NT-6;在异丙基-β-D-硫代半乳糖苷(Isopropy l-βD-th ioga lactos ide,IPTG)诱导的条件下,观察融合蛋白在大肠杆菌JM 109中的表达情况;利用蛋白回收试剂盒对表达产物进行纯化。结果表明经PCR扩增后,得到一分子量为460 bp左右的片段;含有该片段的重组质粒经电泳及双酶切鉴定表明,所得到的重组质粒即为含有目的片段的pGEX 1-NT-6;与带有pGEX 1-λT质粒的细菌相对照,pGEX 1-NT-6质粒转化的大肠杆菌JM 109,在IPTG诱导下表达了特异性的融合蛋白,该蛋白分子量为41 KD。获得的融合蛋白经凝血酶酶切后,得到了分子量大约为15 KD的NT-6成熟肽。本研究成功构建了人源性NT-6成熟肽编码区基因融合表达载体,并纯化了其表达产物,为研究人源性NT-6的生物学功能奠定了基础。
To get the mature peptide of human-derived neurotrophin-6 （NT-6）, NT-6 gene encoding mature peptide was amplified by PCR, using the NT-6 cDNA that had been cloned as templet. The gene encoding mature peptide of NT-6 gene was cloned into pGEX1-λT plasmid to construct the fusion expression vector. Expression of fusion protein in Escherichia coli was defected after induction by isopropyl β-D-thiogalactoside（IPTG）. The mature peptide of NT-6 was collected with GST fusion protein purifying kit. It was shown that a fragment of 460bp was gained by PCR. With the techniques of double-cleave and electrophoresis, the recombinant vector was identified as pGEX1-NT-6. The recombinant vector pGEX1-NT-6 transformed Escherichia coli expressed fusion protein of 41KD after induction by IPTG. Cleaved by thrombin, the mature peptide of NT-6 was obtained;its molecular weight was about 15KD. The cloning and expression of human-derived NT-6 gene encoding mature protein has provided a basis for further studies on the function and clinica, application of NT-6.
出处
《生物医学工程学杂志》
EI
CAS
CSCD
北大核心
2005年第6期1241-1244,共4页
Journal of Biomedical Engineering
基金
四川大学华西基础医学与法医学院分子生物学开放实验室客座研究基金资助
