摘要
将重组质粒 pTXB NT3(6 9kb)与载体 pJLA50 3(4 9kb)分别用Nde 1/BamH 1双酶切 ,然后将前者的人神经营养因子 3 内蛋白子 几丁质结合区 (hNT3 in tein CBD)DNA片段插入后者载体中 ,构建成一个含内蛋白子的温度诱导型表达载体pLZY0 1。经转化后 ,工程菌E coliBL2 1/ pLZY0 1在LB培养基中培养 ,表达产物约为4 2kD的hNT3 intein CBD融合蛋白 ,经DTT不完全化学拆除后 ,可得约 15kD的hNT3,2 7kD的intein CBD及 4 2kD的融合蛋白 ,产物经鸡胚背根神经节测定其生物活性 ,融合蛋白在 80ng ,hNT3在 2 5ng时能较好地促进神经纤维的伸长。
The DNA fragment of human neurotrophic factor 3 intein chitin binding domain (hNT3 intein CBD) in pTXB hNT3 digested with Nde I/BamH I was inserted into MCS of pJLA503, an expression vector by temperature induction, with the same enzyme digestion. A new recombinant plasmid containing hNT3 intein CBD gene fragment, named pLZY01, was constructed. After transferring pLZY01 into the host E.coli BL21, the engineering strain, E.coli BL21/pLZY01 was cultured in LB+Amp medium to A 600nm =0.8 at 37℃, 200r/min and then continuously incubated at 42℃, 3h for induction. It is showed that a fusion protein equal to 42kD was obviously occurred from the results of the culture on SDS PAGE. Furthermore, it is also pointed out that there are 3 bands at 42kD(hNT3 intein CBD), 27kD(intein CBD) and 15kD(hNT3) on SDS PAGE after DTT incomplete reduction. After denaturation and renaturation of inclusion bodies, a pure product at 15kD(hNT3) was obtained on SDS PAGE through affinity chromatography on chitin beads and in term, DTT cleavage. By the preliminary assay of their biological activity toward the dorsal root ganglion of chicken embryo, it is indicated that the products, both fusion protein or target protein(hNT3) have the biological activity at the concentration of 80ng and 25ng respectively.
出处
《高技术通讯》
EI
CAS
CSCD
2000年第3期1-4,共4页
Chinese High Technology Letters
基金
山西省自然科学基金
留学基金项目!( 9710 3 0 )
关键词
人
神经营养因子-3
内蛋白子
温度诱导
融合表达
pTXB hNT3, Human neurotrophic factor 3, Intein,Temperature induction, Fusion expression, Biological activity
