论文部分内容阅读
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p ET28a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。
Objective To prepare the recombinant plasminogen activator (Pla) of Yersinia pestis and lay a foundation for the study on the interaction between Y. pestis and the immunological diagnosis of plague. Methods The pla gene was amplified from the genome of Yersinia pestis using PCR and cloned into p ET28a expression vector. The recombinant plasmid was transformed into E. coli BL21 and induced by IPTG. The expression of the pla gene was detected by SDS-PAGE electrophoresis. The inclusion body Denatured, gradient dilution dialysis method renaturation, SDS-PAGE electrophoresis and Western blot detection of the target protein; milk powder plate assay of plasminogen activator activity. RESULTS AND CONCLUSIONS The agarose gel electrophoresis and sequencing proved that the constructed expression plasmid was successfully constructed. SDS-PAGE electrophoresis indicated that the expression of the protein is in the form of inclusion bodies, and the expression product is mainly the target protein, and the expression level of the target protein is high. Denaturation, renaturation obtained electrophoretic pure Pla protein, and plasminogen activator activity. This study provides a simple, rapid and efficient method for the preparation of active Pla proteins.