感染诱导猪肺炎支原体168株差异表达蛋白的筛选及鉴定

发布时间：2018-04-05 04:06

本文选题：猪肺炎支原体168株　切入点：双向电泳　出处：《山西农业大学》2015年硕士论文

【摘要】：[目的]猪肺炎支原体(Mycoplasma hyopneumoniae, Mhp)是引起猪支原体肺炎Mycoplasma pneumoniae of Swine, MPS)的主要病原。毒力相关因子在支原体的感染与致病中起着十分重要的作用,但目前对毒力相关因子了解甚少。因此,本研究通过对感染猪气管上皮细胞(STEC)诱导前后猪肺炎支原体168强、弱毒株进行差异蛋白质组学分析,试图筛选到一些可能的毒力相关因子。[方法]本试验利用Mhp168强、弱毒株体外感染猪气管上皮细胞(STEC),根据上清支原体含量来筛选出适宜的感染条件。将感染STEC细胞前后的Mhp168强、弱毒株蛋白分别进行双向电泳分离,利用PDQuest V 8.0软件对扫描所得的双向电泳图进行分析(T检验法,差异1.5倍),筛选出差异蛋白。并将筛选出的差异蛋白甘油醛-3-磷酸脱氢酶进行Western-blot验证及延伸因子TU进行了原核表达。[结果]1.根据Mhp168强、弱毒株感染STEC细胞上清支原体量筛选出的感染条件为：感染剂量为1×108CCU,感染时间为48h。2.从感染诱导前后的Mhp168强毒株蛋白双向电泳图谱中筛选出了7个差异的支原体蛋白,其中3个只出现在感染组中,4个在感染后表达上调；从感染诱导前后的Mhp168弱毒株蛋白双向电泳图谱中筛选出了6个差异的支原体蛋白,其中1个只出现在感染组中,4个在感染后表达上调。将筛选出的差异蛋白甘油醛-3-磷酸脱氢酶进行Western-blot验证,结果显示该蛋白在感染诱导后表达上调(p0.01)。3.文章成功对筛选出的差异蛋白EF-TU进行了原核表达,并将表达蛋白进行了Western-blot验证,结果表明表达蛋白能够与抗Mhp阳性血清发生反应,显示具有良好的反应原性。
[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.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.62

【参考文献】