BPI_(23)-haFGF融合蛋白在毕赤酵母X-33中的分泌表达及活性鉴定

Secretion Expression of BPI_(23)-haFGF fusion gene in Pichia pastoris X-33 and Identification of Its Biological Activity

将BPI23-haFGF融合基因克隆到酵母表达载体pPICZαA中,构建出含BPI23-haFGF融合基因的重组质粒pPICZαA-BPI23-haFGF。通过电击将经SacⅠ酶切线性化的pPICZαA-BPI23-haFGF质粒转化到巴斯德毕赤酵母X-33菌中,并通过抗性与表型筛选、PCR鉴定获得高效的工程菌。收集表达上清进行SDS-PAGE和Western blot分析,结果表明,在43KD有与预期大小相符的条带,占上清总蛋白的50%以上。亲和层析纯化后的融合蛋白BPI23-haFGF纯度约90%,体外活性实验结果,纯化产物具有杀死革兰氏阴性菌及促进NIH3T3细胞增殖的双重功能。

The BPI23-haFGF fusion gene was subcloned to the yeast expression vector pPICZaA and the recombinant plasmid pPICZaA-BPI23-haFGF was constructed. After linearization by sac I , the construct was introduced into X-33 yeast cells. The efficient engineering strain was obtained by the resistance and phenotype selection and identi-fied by specific PCR. SDS-PAGE and Western blot analysis indicated that a 43 KD protein band coincident with the anticipated fusion protein size expressed in the culture supernatant of the transformed yeast cells, which accounted for above 50 % of the total proteins of the culture supernatant. About 90 % purity of recombinant BPI23-haFGF fusion protein was obtained by affinity chromatography. The in vitro bioactivity testing showed that the purified fusion protein killed E. coli and promoted proliferation of NIH3T3 cells, suggesting that the recombinant BPI23-haFGF fusion protein possessed both of BPI and FGF functions.