纤维连接蛋白细胞结合区功能多肽在杆状病毒表达系统中的表达与纯化

目的：利用Bac-to-Bac杆状病毒表达系统表达纤维连接蛋白（FN）细胞结合区功能多肽（CBD）,并对其进行纯化和鉴定。方法：经PCR获得人血浆FN-CBD基因,酶切后定向克隆到T载体上,经测序正确后插入pFastBacHTB载体,转化大肠杆菌DH10Bac感受态细胞;用抗生素平板筛选重组杆粒,脂质体介导重组杆粒转染sf9昆虫细胞并进行蛋白表达;经Ni-NTA层析柱对重组多肽进行纯化,对纯化的多肽行SDS-PAGE和Western-blot分析。结果：得到融合6个组氨酸残基的FN-CBD,SDS-PAGE显示其相对分子质量约为36000,Western-blot表明该多肽能与FN的多克隆抗体结合。结论：利用Bac-to-Bac杆状病毒表达系统能成功表达出人血浆FN-CBD,且表达产物具有良好的免疫原性,为后续结构、功能研究奠定了基础。

Expression and Purification of Fibronectin Cell Binding Domain Functional Polypep-tide in Baculovirus Expression System

Objective：To express and purify the recombinant human fibronectin（FN） cell binding domain（CBD） functional polypeptide in Bac-to-Bac baculovirus expression system.Methods：The genes of FN-CBD was amplified by PCR and inserted into pGEM-T vector.After sequenced,the genes was inserted into pFastBacHTB donor plasmid,then the correct recombinant plasmid was further transformed into E.coli DH10Bac competent cells.The recombinant bacmid was screened by LB agar plate containing three kind of antibiotics.Fresh sf9 insect cells were transfected with the recombinant bacmid via lipofectin and the polypeptde was expressed.The recombinant polypeptide was purified with Ni-NTA agarosebead column,and then was further confirmed by SDS-PAGE and Western-blot.Results：The recombinant FN-CBD was successfully expressed in Bac-to-Bac baculovirus expression system.The result of SDS-PAGE showed that the Mr of target polypeptide was 36 000,The result of Western-blot showed that the target polypeptide can react with polyclonal antibodies of FN specially.Conclusion：The recombinant polypeptide can be expressed in sf9 insect cells successfully,it may provide a good strategy for further study the structure and function of the FN-CBD.