大鼠周围神经43kDa蛋白的生化初步分析

The preliminary biochemical analysis of protein 43kDa in the peripheral nerve of rat

目的:提取大鼠损伤坐骨神经组织中表达增强的43kDa蛋白(PNP-43),并对PNP-43进行生化初步分析。方法:纯化抗PNP-43单克隆抗体,将纯化的抗PNP-43单克隆抗体与CNBr-activatedSepharose4B交联,用亲和层析的方法,提取PNP-43,并将提取的PNP-43进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和等电聚焦凝胶电泳(IEF)分析,考马斯亮蓝染色,取SDS-PAGE后染色的43kDa蛋白区带胶条,进行基质辅助激光解吸/电离飞行时间质谱检测(MALDI-TOF-MS),测定肽质量指纹图谱,在相应的蛋白质谱数据库(MS-Fit)中检索。结果:⑴、经层析系统紫外检测,亲和层析后洗脱,在280nm波长处显示一个的紫外吸收峰。⑵、亲和层析获得的蛋白,经SDS-PAGE分析,在43.0kDa标准蛋白位置出现单一蛋白条带。⑶、经IEF分析,PNP-43的等电点(pI)为5.8。⑷、经MALDI-TOF-MS检测,测定肽质量指纹图谱,通过MS-Fit检索未发现有与该蛋白完全相同的质谱数据。结论:用亲和层析的方法,获得了大鼠损伤坐骨神经组织中表达增强的PNP-43,通过质谱分析和MS-Fit检索提示未发现有与该蛋白完全相同的质谱数据。为进一步深入研究PNP-43的结构与功能提供了科学依据。

Objective: To study the peripheral nerve protein 43kDa (PNP-43) i n injured sciatic nerve of rats. Methods:The anti-PNP-43 monoclonal antibody was purified and coupled with CNBr-activated Sepharose 4B. By affinity chromatograp hy, the PNP-43 in injured sciatic nerve of rats was purified, then it was analyz ed by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) and is oelectric focusing(IEF). The PNP-43 was detected by matrix-assisted laser desorp tion/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) and determined by mass mapping. The primary partial structure of PNP-43 was identified by searchi ng the protein database. Results: ⑴.One ultraviolet absorption peak was obtaine d at 280nm when the elution collected by affinity chromatography detected. ⑵.A single protein band was appeared at 43.0kDa when the elution was analyzed by SDS-PAGE. ⑶.The isoelectric point(pI) of PNP-43 analyzed by IEF was about 5.8. ⑷. The mass mapping of peptide was identified by MALDI-TOF-MS, and it was searched by the MS-Fit database that there were not the same protein detabase with PNP-43 . Conclusion: The PNP-43 of rats was obtained by affinity chromatography. The ma ss mapping of the peptide was identified by MALDI-TOF-MS, there were not the sam e proteins in database. This study provides a basis for researching its structur e and functions.