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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 recombinant plasmid pTXB NT3 (69kb) and the vector pJLA503 (49kb) were double digested with Nde1 / BamH1, respectively, and then the former human hTNT3intein CBD ) DNA fragment was inserted into the latter vector to construct an inulin-containing temperature-inducible expression vector pLZY0 1. After transformation, the engineered bacteria E coliBL2 1 / pLZY0 1 were cultured in LB medium to express about 42 kD of hNT3 intein CBD fusion protein, after incomplete chemical removal by DTT, about 15 kD of hNT3, 27 kD intein CBD and 4 2kD fusion protein. The bioactivity of the product was determined by chick embryo dorsal root ganglion. The fusion protein at 80ng and hNT3 at 25ng could promote the nerve fiber elongation.