家蝇幼虫血淋巴蛋白组及血淋巴中热稳定蛋白研究

Study on Proteomics and the Thermostable Protein of Housefly Larvae Hemolymph

目的初步研究家蝇幼虫血淋巴蛋白组学特征及其中的热稳定蛋白。方法使用破壁法提取三龄幼虫血淋巴原液,设置全血淋巴组、沸水浴处理组,采用不同pH范围双向电泳胶条,分析血淋巴中蛋白的pH、分子量分布特点,采用二级质谱对蛋白点进行分子鉴定。结果家蝇幼虫血淋巴中蛋白的分子量主要分布在10kDa～66kDa之间,采用pH3～10的IPG胶,检测到286个蛋白点,采用pH4～7的IPG胶,测到601个蛋白点;血淋巴经沸水浴处理后,检测到41个蛋白点,分子量低于30kDa,比未处理组减少了93.1%,对其中11个蛋白点进行质谱分析,鉴定出4个功能蛋白,包括存储蛋白、气味结合蛋白、铁蛋白重链样蛋白和铁蛋白2轻链蛋白同系物。结论双向电泳能很好地分析家蝇幼虫血淋巴中的蛋白,血淋巴中的蛋白等电点主要分布在pH4～7之间;血淋巴经沸水浴处理后可去掉大量变性蛋白,其上清中可能包含耐热性较好的功能蛋白。

Objective To study the two-dimensional gel electrophoresis （2-DE） characteristics of the hemolymph of Musca domestica larvae. Methods The hemolymph of housefly larvae were collected by scarfskin breaking down, and the whole hemolymph group as well as boiling water bath heating group were set up. The protein pH and molecular weight distri- bution of the hemolymph were analyzed with different pH range immobiline drystrip, and the protein spots were identified with the second order MS. Results The molecular weight range of Musca domestica larvae hemolymph protein was 10 kDa -66 kDa. There were 286 protein spots with pH3 - 10 drystrip and 601 protein spots with pH4 -7 drystrip. After heating by boiling water bath, there were 41 protein spots identified from the hemolymph. The protein spots reduced 93.1% com- pared with that of the unheating group. Mass spectrometry revealed 4 functional proteins within these protein, which are hex- amerin, odor binding protein 2, putative ferritin heavy chain-like protein and ferritin 2 light chain homolog. Conclusion The 2-DE could be used to analyze the protein of the hemolymph of Musca domestica larvae. The isoelectfie point range of the protein in hemolymph is mainly pH4 - 7 ; we could get rid of most metamorphic proteins by boiling water bath, and some heat-resistant protein may remain in the supernatant.