幽门螺杆菌金属蛋白酶原核表达载体的构建与重组表达及纯化

Construction of a prokaryotic expression vector, recombinant expression, and purification of Helicobacter pylori metalloprotease

目的构建幽门螺杆菌(Helicobacter pylori, Hp)金属蛋白酶(metalloprotease, Mtp)基因mtp与麦芽糖结合蛋白基因mbp融合表达载体,诱导Mtp重组表达并纯化表达产物,为Hp致病机制和疫苗研究奠定基础。方法应用PCR从Hp郑州分离株MEL-Hp27的DNA扩增mtp基因,经纯化回收后与克隆载体pMD19-T连接,并进行测序验证。对重组质粒pMD19-T-mtp双酶切,酶切目的片段克隆至表达载体pMAL-c2X,然后用重组质粒pMAL-mtp转化大肠埃希菌(E.coli TB1)。从大肠埃希菌重组子中提取重组质粒,进行酶切和测序鉴定。用分光光度计测定重组菌的生长曲线。用IPTG诱导mtp表达,用SDS-PAGE法分析表达产物,并用Amylose树脂预装柱纯化Mtp蛋白。结果 PCR扩增的mtp基因片段长度为621 bp,重组菌株的酶切和测序鉴定正确;重组菌生长曲线显示重组质粒的转入对受体菌的生长无显著影响;Mtp诱导表达和纯化产物的SDS-PAGE显示,Mtp表达产物为相对分子质量为66.4×10^3的水溶性融合蛋白(rMtp),纯化产物的纯度约为85.6%。结论成功构建mtp基因与麦芽糖结合蛋白mbp基合表达载体,并纯化制备了重组蛋白,为探索Mtp在Hp感染发病机制和抗Hp感染疫苗研究奠定了基础。

Objectives To construct an expression vector for the fusion of the metalloprotease gene of Helicobacter pylori and the gene coding for maltose binding protein,to express the recombinant,and to purify the expression product in order to lay the foundation for development of vaccines and diagnostic antigens.Methods The mtp gene of Helicobacter pylori strain MEL-HP27 was obtained via amplification with PCR.The target fragment was purified,ligated into the cloning vector pMD19-T,and then subjected to nucleotide sequencing.The recombinant plasmid pMD19-T-mtp was digested with restriction enzymes,and the mtp gene was inserted into the expression vector pMAL-c2 X.The recombinant plasmid pMAL-mtp was transformed into E.coli TB1.The recombinant plasmid was isolated from the positive recombinants and identified using restriction enzyme digestion and sequencing.The growth curves were determined using a spectrophotometer.Expression of the recombinant was induced with isopropyl thiogalactoside(IPTG),expression products were analyzed with SDS-PAGE,and the level of expression of the target protein was determined using the software Bandscan5.0.The soluble recombinant Mtp protein was purified using an amylose pre-packed column.Results The PCR product consisted of 621 base pairs.The recombinant bacterium was identified as correctly constructed.The growth curves indicated that the growth of E.coli TB1 was not markedly affected by transferring the recombinant plasmid into the bacterium.SDS-PAGE analysis indicated that the expressed product was a water-soluble recombinant protein(rMtp)with a relative molecular mass of 66.4×10^3.Expression of the recombinant bacterium was induced for 5 h with IPTG(0.3 mmol/L),and the level of expression of the fusion protein represented 28.4%of total bacterial proteins.The purified product had a purity of approximately 85.6%.Conclusions This study is the first to successfully construct an efficient prokaryotic expression system for expression of the fusion of mtp and a gene coding for maltose binding protein.The recombinant protein was purified and prepared,providing an important foundation for investigating the role of Mtp in the pathogenesis of H.pylori-related diseases and developing vaccines against H.pylori infection.