问号钩端螺旋体主要外膜蛋白免疫功能表位及其致炎作用的研究

Immune-functional epitopes and inflammation-inducing effects of the major outer envelope proteins of Leptospira interrogans

目的 :了解问号钩端螺旋体 (简称钩体 )主要外膜蛋白 Omp L 1、Lip L 32和 L ip L 4 1免疫功能表位及其致炎作用。方法 :建立 Ni- NTA亲和层析法 ,提取不同基因型表达的目的重组蛋白 r Omp L1/ 1和 Omp L1/ 2、L ip L32 / 1和 r Lip L32 / 2、Lip L4 1/ 1和 r Lip L4 1/ 2。采用 SDS- PAGE检测上述目的重组蛋白的表达情况和提取物纯度。分别采用 Signal P3.0预测服务器 Signal P- NN软件、Propred MHC class- binding peptide prediction- Pro Pred预测服务器 EMBOSS软件 ,对上述蛋白的信号肽、MHC- 类分子结合肽和 B细胞表位进行分析。以人脐静脉内皮细胞株EVC- 30 4为效应细胞 ,采用 EL ISA检测上述目的重组蛋白诱导人脐静脉内皮细胞 EVC- 30 4分泌 IL - 1、IL - 8和TNF- α的作用。结果 :在 IPTG诱导下 ,所构建的原核表达系统可有效表达 r Omp L1/ 1和 r Omp L1/ 2、r Lip L32 / 1和r Lip L32 / 2、r L ip L4 1/ 1和 r Lip L4 1/ 2 ,其产量分别约占细菌总蛋白的 30 %和 15 %、4 0 %和 35 %、15 %和 10 %。提纯后的目的重组蛋白 SDS- PAGE后均仅见单一的蛋白条带。Omp L1s、Lip L32 / 1和 Lip L32 / 2、Lip L4 1s的信号肽分别位于 N端 1- 2 4、1- 2 1和 1- 2 4、1- 2 4位氨基酸残基。 Omp L 1s。

Objective: To investigate the immune functional epitopes and inflammation inducing effects of the major outer envelope proteins of Leptospira interrogans. Methods: Ni NTA affinity chromatography was established to extract the target recombinant proteins rOmpL1/1 and OmpL1/2,LipL32/1 and rLipL32/2,LipL41/1 and rLipL41/2 expressed by the different genotypes.By using Signal P NN software in Signal P3.0 prediction server, EMBOSS software in propred MHC class Ⅱ binding peptide prediction ProPred prediction server,the possible signal peptides,MHC Ⅱ binding peptides and lymphocyte B epitopes were analyzed.The IL 1,IL 8 and TNF α secretion in human umbilical vein endothelial cell line EVC 304 induced by target recombinant proteins were measured by ELISA. Results: Under the inducement of IPTG,the constructed prokaryotic systems efficiently expressed rOmpL1/1 and rOmpL1/2,rLipL32/1 and rLipL32/2,and rLipL41/1 and rLipL41/2 with outputs of 30% and 15%,40% and 35%,and 15% and 10% of the total bacterial proteins,respectively.Each of the purified target recombinant proteins showed a single protein band in SDS PAGE.The signal peptides of OmpL1s,LipL32/1 and LipL32/2 ,and LipL41s were located at the N ends of 1-24,1-21 and 1-24,and 1-24 amino acid residuals, respectively .OmpL1s,LipL32s and LipL41s displayed 2,2 and 1 same major epitopes of MHC Ⅱ binding peptides and lymphocyte B and OmpL1/2 had another one (59-78).The different dosages of rOmpL1s, rLipL32s and rLipL41s increased the secretion of IL 1α,IL 8 and TNF α ( P <0.05) in EVC 304 cells.The IL 1α levels reached the highest at the 24 h and then declined,while the IL 8 and TNF α levels after 48 h treatment were higher that those after 24 h. Conclusion: The expression products in ompL1/1 , lipL32 or lipL41 genotypes of L.interrogans contain similar immune functional epitopes.rOmpL1/1 and rOmpL1/2,rLipL32/1 and rLipL32/2,and rLipL41/1 and rLipL41/2 are able to directly induce inflammatory reaction in EVC 304 cells.