【摘要】：目的對(duì)斯氏并殖吸蟲(Paragonimus skrjabini)囊蚴、童蟲和成蟲蛋白質(zhì)組進(jìn)行研究。方法在貴州省斯氏并殖吸蟲疫源地開陽(yáng)、畢節(jié)采集溪蟹,從中分離囊蚴置于PBS中,灌胃感染SD大鼠3只(10個(gè)囊蚴/只),喂食感染雄性犬3只(20個(gè)囊蚴/只)。感染大鼠后1個(gè)月解剖取童蟲,感染犬后3個(gè)月解剖取成蟲。將囊蚴、童蟲和成蟲中加入中性組織裂解液,冰上超聲破碎提取總蛋白,Bradford法測(cè)定蛋白含量并進(jìn)行十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳(SDS-PAGE)及雙向凝膠電泳,采用PDquest 8.0軟件對(duì)雙向凝膠電泳圖像進(jìn)行分析尋找差異點(diǎn)。對(duì)差異點(diǎn)進(jìn)行酶解、質(zhì)譜鑒定。最后進(jìn)行NCBI在線及本地?cái)?shù)據(jù)庫(kù)檢索。結(jié)果 SDS-PAGE顯示囊蚴、童蟲和成蟲總蛋白主要集中分布在相對(duì)分子質(zhì)量(Mr)25 000~116 000。雙向凝膠電泳共篩選出51個(gè)差異蛋白點(diǎn),分別為囊蚴20個(gè)、童蟲25個(gè)、成蟲6個(gè)。因成蟲差異點(diǎn)灰度值兩兩間的比值變化遠(yuǎn)小于囊蚴與童蟲,故未進(jìn)行質(zhì)譜鑒定。36個(gè)囊蚴和童蟲的肽段序列進(jìn)行分析后發(fā)現(xiàn),在線檢索蛋白種類主要為無色桿菌(Achromobacter lyticus)蛋白酶Ⅰ(一種賴氨酸特異性的絲氨酸酶)、脫氫抗壞血酸還原酶蛋白、谷胱甘肽S轉(zhuǎn)移酶DHAR2類似物、熱激蛋白Hsp82及Hsp96-β、肌動(dòng)蛋白和半胱氨酸酶抑制劑等;本地復(fù)殖目數(shù)據(jù)庫(kù)檢索主要蛋白種類是半胱氨酸酶、肌動(dòng)蛋白和熱激蛋白。結(jié)論斯氏并殖吸蟲囊蚴與童蟲蛋白組成有差異,囊蚴含有肌動(dòng)蛋白,童蟲含有解毒及應(yīng)激蛋白,兩者都含有水解酶類及半胱氨酸酶。
[Abstract]:Objective to study the proteome of paragonimus skrjabini (Paragonimus skrjabini) cysticercus, juvenile worm and adult worm. Methods in Kaiyang, Guizhou Province, where paragonimus skrjabini foci were collected, the cysticercus was isolated and placed in PBS. Three SD rats (10 cysticercaria / cysticercaria) were fed with infected male dogs (20 cysticercaria / rat). The adult worms were dissected 1 month after infection and 3 months after infection. The neutral tissue lysate was added to cysticercus, juvenile worm and adult. The total protein was extracted by ultrasonic crushing on ice. The protein content was determined by SDS-PAGE and two dimensional gel electrophoresis. The two-dimensional gel electrophoresis image was analyzed by PDquest 8.0 software to find out the difference point. The differential sites were hydrolyzed by enzyme and identified by mass spectrometry. Finally, NCBI online and local database retrieval. Results SDS-PAGE showed that the total proteins of cysticercus, juvenile worm and adult were mainly distributed in the relative molecular weight of (Mr) 25 000 ~ 116 000. A total of 51 differential protein spots were screened by two dimensional gel electrophoresis, including 20 cysticercaria, 25 juvenile worms and 6 adult worms. Because the ratio of gray value of the difference point of adult is much smaller than that of cysticercus and child worm, it is not identified by mass spectrometry. After analyzing the peptide sequence of 36 cysticercus and child worm, it was found that, The online search proteins are mainly (Achromobacter lyticus) protease I (a lysine-specific serine enzyme), dehydroascorbic acid reductase protein and glutathione S-transferase DHAR2 analogue. Heat shock protein (Hsp82) and Hsp96- 尾, actin and cysteinase inhibitor were found in the local colonization database, and the main proteins were cysteinase, actin and heat shock protein. Conclusion there are differences in protein composition between metacercaria of paragonimus skrjabini and that of child worm. Actinomycin is found in cysticercaria and detoxification and stress protein in juvenile worm. Both of them contain hydrolases and cysteinase.
【作者單位】： 貴州省疾病預(yù)防控制中心;貴州師范大學(xué)生命科學(xué)院;
【基金】：貴州省優(yōu)秀科技教育人才省長(zhǎng)資金項(xiàng)目(No.2010-91) 貴州省科學(xué)技術(shù)基金(No.[2012]2210)~~
【分類號(hào)】：R383.23

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