结缔组织病肺间质病相关自身抗原人杀菌/渗透增强蛋白B1蛋白原核表达体系的建立

Establishment of prokaryotic expression system of autoantigen human bactericidal/permeability-incre-asing fold-containing B1

目的建立肺间质病相关自身抗原人杀菌/渗透增强蛋白B1(BPIFB1)的原核表达体系,为该蛋白的免疫学研究提供工具。方法以pGEM-C20ORF114重组质粒为模板,采用特异引物扩增BPIFB1基因编码区,克隆入pET28a-MBP-His和pGEX-5X-1载体中。重组的pET-BPIFB1-MBP-His和pGEX-BPIFB1-GST质粒转染Top10感受态细胞;鉴定阳性克隆,并选取测序正确的质粒转化Rosetta(DE3)蛋白表达菌株;异丙基硫化半乳糖苷(IPTG)诱导表达,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)及蛋白质印迹法检测重组融合蛋白的表达,并用尿素变性复性和镍树脂亲和层析柱纯化目的蛋白。结果PCR成功扩增出特异条带,符合BPIFB1基因大小;经双酶切克隆至表达载体,测序证实重组质粒pET-BPIFB1-MBP-His和pGEX-BPIFB1-GST构建成功。SDS-PAGE检测BPIFB1-MBP与BPIFB-GST融合蛋白均主要以包涵体形式表达。蛋白质印迹法检测,重组BPIFB1-MBP-His蛋白能被抗6×His抗体识别。经尿素变性,镍柱亲和层析纯化,然后蛋白复性,获得纯化的可溶性BPIFB1-MBP融合蛋白。结论通过构建重组质粒pET-BPIFB1-MBP-His,并采用变性条件下先镍柱亲和纯化后再复性的方法,成功建立BPIFB1蛋白原核表达体系。

Objective To establish a prokaryotic expression system of interstitial lung disease associated autoantigen human bactericidal/permeability-increasing fold-containing B1(BPIFB1),providing tools for the study on its function in immune responese.Methods The coding region of BPIFB1 gene was amplified with specific primers from recombinant pGEM-C20ORF114 plasmid and cloned into the pET28a-MBP-His and pGEX-5X-1 vectors.The recombinant pET-BPIFB1-MBP-His and pGEX-BPIFB1-GST plasmids were transfected into Top10 cells.The positive clones were selected and sequenced.The correct clones of pET-BPIFB1-MBP-His and pGEX-BPIFB1-GST were transfected into prokaryotic expression strain Rosetta(DE3)and induced by Isopropylβ-D-Thiogalactoside(IPTG).The expression of recombinant BPIFB1 fusion protein was analyzed by SDS-PAGE and Western blotting,and purified by urea modified and renaturation and affinity chromatography of nickel NTA-resin.Results The polymerase chain reaction(PCR)produced specific product with the molecular weight equivalent to that of BPIFB1.The recombinant pET-BPIFB1-MBP-His and pGEX-BPIFB1-GST plasmids were cloned by double restriction enzyme digestion and ligation and confirmed by sequencing.The SDS-PAGE result showed that both BPIFB1-MBP and BPIFB1-GST fusion proteins were mainly expressed in the form of inclusion bodies.The Western blotting result revealed that the recombinant BPIFB1-MBP-His protein could be recognized by Anti-6×His antibody.The purified soluble BPIFB1-MBP fusion protein was obtained by urea denaturation,affinity chromatography of nickel NTA-resin and then renaturation after purification.Conclusion The BPIFB1 prokaryotic expression system is established by construct recombinant plasmid pET-BPIFB1-MBP-His,and an approach of renaturation after nickel resin affinity purification in denatured condition.