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目的探讨蠕形螨全蛋白提取方法,初步分析蠕形螨蛋白组分。方法采用自制裂解液和RIPA裂解液分别提取蠕形螨全蛋白,通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分离比较蛋白提取效果,用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)鉴定部分蛋白质条带,Mascot检索软件查询NCBInr数据库,对蠕形螨蛋白组分进行初步分析。结果自制裂解液提取蠕形螨蛋白的效果优于RIPA裂解液。经数据库检索比对出21种可信蛋白,其中170 kD条带比对出的蛋白为肌动蛋白和胞质肌动蛋白,130 kD条带比对出的蛋白为谷氨酰胺连接酶和腺苷酰转移酶;15 kD以下条带比对出的蛋白大部分为组蛋白。结论自制裂解液液氮研磨法可提取到较高质量的蠕形螨全蛋白,实验提取并分析了蠕形螨的蛋白质组成,为蠕形螨蛋白组学研究奠定基础。
Objective To investigate the extraction method of total protein from Demodex mites and to analyze the protein components of Demodex mites. Methods The total protein of Demodex mite was extracted by homemade lysate and RIPA lysate. The protein extraction was compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the protein was extracted by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) to identify part of the protein bands, Mascot search software query NCBInr database for preliminary analysis of Demodex protein components. Results The extraction of Demodex protein by homemade lysate was better than RIPA lysate. Twenty-one kinds of authentic proteins were identified by database search. Among them, the protein of 170 kD bands was actin and cytoplasmic actin, and the protein of 130 kD bands was glutamine ligase and gland Glycosyltransferase; most of the proteins in the bands below 15 kD were histones. Conclusion The high quality Demodex mite protein can be extracted by liquid nitrogen grinding method. The protein composition of Demodex mite was extracted and analyzed experimentally, which laid the foundation for proteomics of Demodex mite.