人β-NGF在大肠杆菌中的表达、纯化及活性测定

Purification and Biological Activity of Recombinant Human β-NGF Expressed in E. coli

目的利用大肠杆菌表达系统表达经密码子突变的重组人神经生长因子(rhβ-NGF),并对表达产物进行分离纯化和生物学活性鉴定。方法利用化学合成法合成经密码子突变后的人神经生长因子β亚基成熟肽段基因及前导肽基因;构建原核表达载体pET28a(+)+[rhβ-NGF],转化大肠杆菌株BL21(λDE3),获得基因工程菌;利用IPTG诱导rhβ-NGF在基因工程菌中表达并收集菌体;分离纯化包涵体蛋白,经体外复性及肠激酶切割去除前导肽后进一步通过镍柱亲和色谱获得rhβ-NGF;通过PC12细胞存活法检测rhβ-NGF的活性。结果构建的基因工程菌能大量表达包涵体蛋白,体外稀释复性后获得的重组蛋白经SDS-PAGE电泳检测其纯度大于95%,Western blot检测其为目标蛋白;重组蛋白经肠激酶切割及进一步分离纯化后得到的目的蛋白经SDS-PAGE电泳检测为单一条带,纯度大于99%;经Western blot鉴定其为rhβ-NGF;生物学活性检测结果显示其比活性约为500 000 u·mg-1。结论通过优化设计密码子,建立重组人神经生长因子大肠杆菌表达系统,并实现其在大肠杆菌中的高水平表达。

OBJECTIVE To express the recombinant human nerve growth factor by using codon mutation of Escherichia coli （ rh β- NGF） ,separate and purify the expression products, and determine the biological activity. METHODS The mature peptide gene and propeptide gene of rh β-NGF, whose genetic codon were mutated to prokaryotic preference codon, were chemically synthesized, and a his tag gene was fused with them. Then, the fusion gene was inserted into pET28a（ ＋ ） expression vector, and the recombinant plasmid was transferred into Escherichia coli BL21 to construct high expression genetic engineering bacteria of rh β-NGF. Upon induction with isopropyl β-D-thiogalactoside （IPTG） , the fusion protein was expressed in Escherichia coli BL21 cells, and large amounts of recombinant protein accumulated in intracellular inclusion bodies. Inclusion bodies were isolated and purified from the BL21 cells and solubilized in 8M urea. Then fusion protein was produced by in vitro refolding and purified by Ni-chelating affinity chromatography, and the propeptide was cut out by enterokinase enzyme. Finally, the purified rh β-NGF was identified by SDS-PAGE and Western blot, and its biological activity was analyzed by PC12 cell culture method. RESULTS Inclusion body protein was expressed in a large amount in the gene engineering bacteria constructed in vitro. The purity of the diluted refolded recombinant protein was greater than 95% as shown by SDS-PAGE electrophoresis, which was proved to be the target protein by Western blot. The recombinant protein was treated by enterokinase cleavage and further separation and purification to obtain the target protein which displayed as a single band in SDS- PAGE electrophoresis with purity of more than 99% ; the product was identified as rhl3-NGF by Western blot; biological activity tests showed that the specific activity was about 500 000 u · mg- 1. CONCLUSION The genetic codon of rh β-NGF is optimizied, and constructed a high expression genetic engineering bacteria of rh β-NGF. Upon induction with IPTG, large amounts of recombinant protein accumulate in Escherichia coli BL21 cells.