本文選題：豬肺炎支原體168株　切入點：雙向電泳　出處：《山西農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：[目的]豬肺炎支原體(Mycoplasma hyopneumoniae, Mhp)是引起豬支原體肺炎Mycoplasma pneumoniae of Swine, MPS)的主要病原。毒力相關(guān)因子在支原體的感染與致病中起著十分重要的作用,但目前對毒力相關(guān)因子了解甚少。因此,本研究通過對感染豬氣管上皮細(xì)胞(STEC)誘導(dǎo)前后豬肺炎支原體168強(qiáng)、弱毒株進(jìn)行差異蛋白質(zhì)組學(xué)分析,試圖篩選到一些可能的毒力相關(guān)因子。[方法]本試驗利用Mhp168強(qiáng)、弱毒株體外感染豬氣管上皮細(xì)胞(STEC),根據(jù)上清支原體含量來篩選出適宜的感染條件。將感染STEC細(xì)胞前后的Mhp168強(qiáng)、弱毒株蛋白分別進(jìn)行雙向電泳分離,利用PDQuest V 8.0軟件對掃描所得的雙向電泳圖進(jìn)行分析(T檢驗法,差異1.5倍),篩選出差異蛋白。并將篩選出的差異蛋白甘油醛-3-磷酸脫氫酶進(jìn)行Western-blot驗證及延伸因子TU進(jìn)行了原核表達(dá)。[結(jié)果]1.根據(jù)Mhp168強(qiáng)、弱毒株感染STEC細(xì)胞上清支原體量篩選出的感染條件為：感染劑量為1×108CCU,感染時間為48h。2.從感染誘導(dǎo)前后的Mhp168強(qiáng)毒株蛋白雙向電泳圖譜中篩選出了7個差異的支原體蛋白,其中3個只出現(xiàn)在感染組中,4個在感染后表達(dá)上調(diào)；從感染誘導(dǎo)前后的Mhp168弱毒株蛋白雙向電泳圖譜中篩選出了6個差異的支原體蛋白,其中1個只出現(xiàn)在感染組中,4個在感染后表達(dá)上調(diào)。將篩選出的差異蛋白甘油醛-3-磷酸脫氫酶進(jìn)行Western-blot驗證,結(jié)果顯示該蛋白在感染誘導(dǎo)后表達(dá)上調(diào)(p0.01)。3.文章成功對篩選出的差異蛋白EF-TU進(jìn)行了原核表達(dá),并將表達(dá)蛋白進(jìn)行了Western-blot驗證,結(jié)果表明表達(dá)蛋白能夠與抗Mhp陽性血清發(fā)生反應(yīng),顯示具有良好的反應(yīng)原性。
[Abstract]:[objective] Mycoplasma hyopneumoniae (Mhp) is the main pathogen of Mycoplasma pneumoniae of swine pneumonia.Virulence related factors play an important role in mycoplasma infection and pathogenicity, but little is known about virulence related factors.Therefore, this study tried to screen some possible virulence related factors by differential proteomics analysis of mycoplasma pneumoniae 168 strong and attenuated strains before and after induction of STEC-infected swine trachea epithelial cells (STECs).[methods] in this experiment, we used Mhp168 strong and attenuated strain to infect pig trachea epithelium in vitro, and screened the suitable infection condition according to the content of supernatant mycoplasma.The Mhp168 strong and attenuated strains were separated by two dimensional electrophoresis before and after infection with STEC cells. The scanning results were analyzed by PDQuest V 8.0 software. The difference was 1. 5 times, and the differential proteins were screened out.The differential protein glyceraldehyde-3-phosphate dehydrogenase was confirmed by Western-blot and the extension factor TU was expressed in prokaryotic cells.[result] 1.According to the Mhp168 strong, attenuated strain infection STEC cell supernatant mycoplasma quantity screening the infection condition is: the infection dose is 1 脳 108 CCU, the infection time is 48h.2.Seven differentially expressed mycoplasma proteins were screened from the two dimensional electrophoresis patterns of Mhp168 virulent proteins before and after infection induction, 3 of which only appeared in the infected group and 4 were up-regulated after infection.Six differentially expressed mycoplasma proteins were screened from the two dimensional electrophoresis patterns of Mhp168 attenuated strain proteins before and after infection induction, one of which only appeared in the infected group and four were up-regulated after infection.The differential protein glyceraldehyde-3-phosphate dehydrogenase was identified by Western-blot. The results showed that the protein up-regulated the expression of glyceraldehyde -3-phosphate dehydrogenase after infection induction.The differentially expressed protein EF-TU was successfully expressed in prokaryotic cells, and the expressed protein was verified by Western-blot. The results showed that the expressed protein could react with anti-#en2# positive serum and had good reactivity.
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.62

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