人源性抗bFGF单链抗体表达载体的构建与酵母表达

High Level Expression of Human ScFv against bFGF in Pichia pastoris

为了提高人源性抗bFGF抗体的表达量,从噬菌体抗体库筛选出的人源性抗bFGF抗体基因中亚克隆单链抗体(single chain fragment variable,ScFv)基因,并将其构建到酵母表达载体pPICZαA中。表达载体质粒经线性化后,电转化法转化至毕赤酵母GS115中,甲醇诱导表达。表达产物经镍离子亲和层析和阴离子交换层析纯化,并检测其生物学活性。酶切鉴定结果显示人源性的酵母表达载体构建成功。SDS-PAGE和Western blot结果显示,抗bFGF单链抗体获得了高效表达,表达量可达124mg/L,目的蛋白大小为36 kDa左右。通过两步纯化方案,目的蛋白的纯度可达95%以上。ELISA结果显示纯化的目的蛋白可与bFGF特异性结合。CCK8检测结果显示,纯化的抗bFGF单链抗体可剂量依赖性地抑制人肺腺癌细胞株A549的增殖。研究结果表明在毕赤酵母中可获得人源性抗bFGF单链抗体高效表达,表达产物具有很好的生物学活性。

In order to high level express anti-bFGF ScFv antibody in Pichia pastoris,the gene of human anti-bFGF ScFv was subcloned,and constructed into expression vector pPICZαA.The constructed expression vector pPICZαA-ScFv was linearised and transformed to Pichia pastoris by electroporation.The transformants were induced by methanol,and the anti-bFGF ScFv was expressed.The expression products were purified by affinity chromatography of Ni-Seproase 6 FF and ion exchange chromatography of DEAE Sepharose FF.The results of SDS-PAGE and Western blotting showed that anti-bFGF ScFv was high level expressed successfully and the expression quantity was about 124mg/L.The target protein was purified from the expression products and the purity was more than 95 %.The results of ELISA showed that the purified recombinant ScFv could combine with bFGF specifically.The results of CCK8 showed that the purified recombinant ScFv could inhibit the proliferation of A549 cells in a dose-dependent manner in vitro.The results demonstrated that the anti-bFGF ScFv can be high level expressed in Pichia pastoris with good biological activity.