CAJ | 학술논문

目的：制备鼠疫耶尔森菌（Yersinia pestis）重组纤溶酶原激活剂（Pla）蛋白，为研究鼠疫菌蛋白之间相互作用和鼠疫的免疫学诊断奠定基础。方法应用PCR从鼠疫菌基因组中扩增pla基因，将其克隆到pET28a表达载体中；转化大肠杆菌BL21中，IPTG诱导表达；SDS-PAGE电泳检测表达结果；应用8 mol／L尿素将包涵体变性，梯度稀释透析法将其复性，SDS-PAGE电泳和Western印迹检测目的蛋白；奶粉平板法检测纤溶酶原激活剂活性。结果与结论琼脂糖凝胶电泳和测序结果证明表达质粒构建成功；SDS-PAGE电泳结果表明Pla蛋白以包涵体形式表达，表达产物主要为目的蛋白，且目的蛋白的表达量较高；包涵体经变性、复性得到了电泳纯Pla蛋白，并具有纤溶酶原激活剂活性。该研究提供了一种简单、快速、高效制备具有活性的Pla蛋白的方法。
목적：제비서역야이삼균（Yersinia pestis）중조섬용매원격활제（Pla）단백，위연구서역균단백지간상호작용화서역적면역학진단전정기출。방법응용PCR종서역균기인조중확증pla기인，장기극륭도pET28a표체재체중；전화대장간균BL21중，IPTG유도표체；SDS-PAGE전영검측표체결과；응용8 mol／L뇨소장포함체변성，제도희석투석법장기복성，SDS-PAGE전영화Western인적검측목적단백；내분평판법검측섬용매원격활제활성。결과여결론경지당응효전영화측서결과증명표체질립구건성공；SDS-PAGE전영결과표명Pla단백이포함체형식표체，표체산물주요위목적단백，차목적단백적표체량교고；포함체경변성、복성득도료전영순Pla단백，병구유섬용매원격활제활성。해연구제공료일충간단、쾌속、고효제비구유활성적Pla단백적방법。
Objective To prepare recombinant plasminogen activator(Pla) protein in E.coli BL21 cells that can be used in studying interactions between Yersinia pestis proteins and immunologic diagnosis of plague.Methods The pla gene was amplified by PCR and cloned into the pET28a expression vector.E.coli BL21 competent cells were transformed with the recombinant vectors, and isopropyl-β-D-thiogalactopyranoside ( IPTG) was added to induce expression of Pla protein. The expressed protein was detected by SDS-PAGE electrophoresis.The inclusion bodies of Pla protein were denatured in 8 mol/L urea, and then refolded using gradient urea solutions.The purified protein was identified by SDS-PAGE electrophoresis and Western blot.Results and Conclusion The constructed expression vector was demonstrated to be correct through agarose gel electrophoresis and sequencing.The recombinant Pla protein was accumulated as an inclusion body in E.coli, and the overexpression product was mainly a target protein, the yield of which was very high.SDS-PAGE purity of the bioactive Pla protein was obtained by denaturing and refolding the inclusion bodies.This study provides a simple and quick method for highly efficient preparation of biologically active Pla protein.