pQE-hbFGF表达系统的构建及其蛋白质的表达和纯化

 目的　构建pQE-hishbFGF表达载体,通过一步纯化得到6×HishbFGF蛋白质。方法　用PCR扩增目的基因hbFGF ,连接到pQE40载体上,转化到Top10菌株筛选重组子,经测序后将质粒转化到表达菌M15上,经IPTG诱导表达,超声波破菌后通过镍离子螯合层析一步纯化得6×HishbFGF蛋白质,ELISA鉴定产物 ,用MTT法检测产物促细胞增殖的生物活性。结果　hbFGF基因插入pQE载体中,在M15中6×HishbFGF的表达量达到20%,且为可溶性表达。经一步纯化后得到N端带 6个组氨酸的hbFGF蛋白,纯度为96%,6×HishbFGF具有免疫原性及促进NIH3T3细胞增殖的活性。结论　 6×HishbFGF蛋白质在M15中可溶性地高效表达,并经一步亲和层析得到纯度高活性高的产物,降低了生产成本,为大规模生产hbFGF及hbFGF的应用研究奠定了基础。

Construction of expressive system pQE-hbFGF and its expression and purification

OBJECTIVE To construct pQE-hishbFGF expression vector,obtain 6×HishbFGF by single purification step of affinity chromatography.METHODS The hbFGF gene was amplified by polymerase chain reaction, and cloned into pQE40 vector.Recombinant plasmid was sequenced and was transformed to Escherichia coli M15.6×HishbFGF expressed after IPTG induction was purified by Ni-NTA affinity column.Recombinant 6×HishbFGF was identified by ELISA, and its biological activity was determined by MTT test.RESULTS hbFGF gene was cloned into pQE vector.Recombinant protein was accounted for about 20% of total bacterial protein after 4 h of induction.The purity of 6×HishbFGF reached 96% after affinity chromatography.HishbFGF bound with hbFGF antibody and stimulated the proliferation of the NIH 3T3 fibroblasts.CONCLUSION HishbFGF was successfully expressed .High purified recombinant protein was obtained by one affinity column.It reduced the cost of production and underlay the base of large scale production.