卵泡刺激素功能肽与受体胞外区不同片段结合作用的分析  

Analysis of the Binding Effect of Follicle Stimulating Hormone Peptide and Different Fragments of Extracellular Domain

作　　者：赵婷[1] 席欧彦 秦瑞坪 邱玲玲[1] 马晓玲[1] 李江伟[1] 

机构地区：[1]新疆大学生命科学与技术学院新疆生物资源基因工程重点实验室,乌鲁木齐830046

出　　处：《新疆农业科学》2017年第7期1284-1290,共7页Xinjiang Agricultural Sciences

基　　金：国家自然科学基金项目"抗卵泡刺激素受体纳米抗体的制备及其在肿瘤分子显像和抗血管治疗中的作用"(81260333)~~

摘　　要：【目的】卵泡刺激素(FSH)33-53肽可以与FSH受体(FSHR)结合,并激活下游信号,被作为FSH功能肽。然而其在受体上的具体结合位置还不清楚。阐明FSH33-53肽在FSHR上的结合区域,为基于FSH的疫苗设计提供依据。【方法】采用重组的FSHR胞外区(ECD)和富含亮氨酸区(LRR)及合成的FSHR9-30区分析这些FSHR的功能区域与FSH33-53的结合作用。PCR扩增得到h FSHR ECD和h FSHR LRR目的基因,构建重组质粒p ET22b-FSHR-ECD和p ET22b-FSHR-LRR,表达纯化获得蛋白FSHR-ECD和FSHR-LRR。采用多肽合成法获得FSHR9-30-KLH。通过ELISA方法检测受体片段与FSH33-53肽的结合和亲和力。【结果】获得FSHR-ECD和FSHR-LRR蛋白,其相对分子质量(Mr)分别为43和32k Da。在受体为0.5μgm L,配体为2.5μgm L时,三种蛋白均与FSH33-53肽结合。ELISA检测受体与配体的亲和力分别为0.21×10^(-6)、0.45×10^(-6)和0.056×10^(-6)mol/L。【结论】FSHR 9-30片段与FSH33-53肽的结合较其它两个片段结合力强。【Objective】The FSH33-53 peptide binds to its receptor FSHR and activates the downstream signal as a FSH functional peptide.However,its specific binding position on the receptor is unclear.The aim of this study is to elucidate the binding region of the FSH33-53 peptide on the receptor FSHR in the hope of providing a basis for FSH-based vaccine design.【Method】The extracellular domain( ECD) and leucine-rich repeat( LRR) of follicle-stimulating hormone receptor was amplified by polymerase chain reaction( PCR) and constructed recombinant plasmids p ET22b-FSHR-ECD and p ET22b-FSHR-LRR.Protein FSHR-ECD and FSHR-LRR were obtained by expression and purification.FSHR9-30-KLH.was obtained by peptide synthesis,and binding and affinity of receptor fragments with FSH33-53 peptide were detected by ELISA method.【Result】The proteins FSHR-ECD and FSHR-LRR were successfully expressed and purified and their relative molecular mass( MR) was 43 and 32 kda.,respectively.When the receptor was 0.5 μg/m L and the ligand was 2.5 μg/m L,the three proteins were bound to the FSH33-53 peptide.The affinity of ELISA to detect the ligand and ligand was 0.21 × 10-6,0.45 × 10-6and 0.056 × 10-6mol/L,respectively.【Conclusion】The binding of LRR fragment to FSH33-53 peptide is stronger than that of the other two fragments.

关 键 词：FSH33-53肽 LRR FSHR9-30肽 胞外区 亲和力 

分 类 号：S188[农业科学—农业基础科学] Q786[生物学—分子生物学]