CAJ | 학술논문

目的：表达并纯化可溶性重组钩端螺旋体层粘连蛋白结合蛋白Lsa20，并对其与层粘连蛋白（LN）相互作用的生物特性进行分析。方法：优化合成Lsa20基因，连接到表达载体pET28a（+）上，转化大肠杆菌BL21（DE3），IPTG诱导蛋白表达，然后利用Ni-NTA亲和介质纯化目标蛋白，重组蛋白经超滤浓缩后，进行相关ELISA、流式细胞术、间接免疫荧光实验。结果：构建了重组蛋白表达菌株，目标蛋白呈可溶性表达，纯化后纯度达90%以上；ELISA证实重组Lsa20与LN有较强的亲和力，测得解离常数为（27.23±2.53） nmol/L；流式细胞术及间接免疫荧光实验结果表明重组Lsa20可黏附到COS-7细胞表面。结论：重组Lsa20在体外能够与层粘连蛋白结合，具有同天然蛋白类似的生物学特性。同时，以Lsa20与LN相互作用实验为例，建立了研究LN结合蛋白生物功能的基本方案，为后续鉴定和研究新的LN结合蛋白的作用规律及黏附机制奠定了良好的实验基础。
목적：표체병순화가용성중조구단라선체층점련단백결합단백Lsa20，병대기여층점련단백（LN）상호작용적생물특성진행분석。방법：우화합성Lsa20기인，련접도표체재체pET28a（+）상，전화대장간균BL21（DE3），IPTG유도단백표체，연후이용Ni-NTA친화개질순화목표단백，중조단백경초려농축후，진행상관ELISA、류식세포술、간접면역형광실험。결과：구건료중조단백표체균주，목표단백정가용성표체，순화후순도체90%이상；ELISA증실중조Lsa20여LN유교강적친화력，측득해리상수위（27.23±2.53） nmol/L；류식세포술급간접면역형광실험결과표명중조Lsa20가점부도COS-7세포표면。결론：중조Lsa20재체외능구여층점련단백결합，구유동천연단백유사적생물학특성。동시，이Lsa20여LN상호작용실험위례，건립료연구LN결합단백생물공능적기본방안，위후속감정화연구신적LN결합단백적작용규률급점부궤제전정료량호적실험기출。
Objective: To express and purify fusion recombinant leptospirosis laminin-binding protein Lsa20, ana?lyze interaction between Lsa20 and laminin(LN). Methods: The Lsa20 gene was synthesized and subcloned into the prokaryotic expression vector pET28a(+), and the recombinant protein was induced to express in E.coli BL21 (DE3) by IPTG. The expressed recombinant soluble protein was purified by a Ni-Resin column. The laminin-binding characteristics of recombination protein were analyzed by ELISA, flow cytometry and immunofluorescence. Results: Reconstructed Lsa20 was expressed in E.coli BL21(DE3) in soluble form, when purified with Ni-Resin, Lsa20 protein reached more than 90% of total proteins. ELISA based solid-phase binding assays demonstrated that there were high affinity interactions affinity between Lsa20 and LN. The results also revealed the dissociation constants values of (27.23±2.53) nmol/L for the binding of Lsa20 to LN. In addition, flow cytometry and immuno?fluorescence test also suggested that Lsa20 can adhere to surface of COS-7 cell effectually. Conclusion: The re?combinant Lsa20 protein can bind to LN in vitro and display a typical adhesin-like function. This work would lay the foundation for further identification and research of the novel laminin-binding protein.