本文選題：布魯氏菌 + sRNA　； 參考：《華中農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：布魯氏菌是一種革蘭氏陰性兼性胞內(nèi)寄生菌,能夠引起人類和家畜的布魯氏菌病,通過皮膚黏膜、呼吸道和消化道傳播。羊、牛、豬等宿主被感染后,會出現(xiàn)流產(chǎn)和不孕不育等癥狀,而人則會出現(xiàn)關(guān)節(jié)炎、睪丸炎、波浪熱、多汗等特征。布魯氏菌感染機體后,能在機體內(nèi)建立免疫逃避機制。最近研究表明,細(xì)菌中存在一類非編碼的小RNA(small non-coding RNA,s RNA),具有潛在的調(diào)控細(xì)菌代謝以及毒力相關(guān)基因作用。本課題組已有的RNA-seq分析預(yù)測表明,羊種布魯氏菌的s RNA Abc R 1極可能是具有調(diào)控功能的s RNA。布魯氏菌的s RNA靶定其m RNAs然后發(fā)揮轉(zhuǎn)錄后調(diào)控作用。通過查閱文獻(xiàn)同源性比對以及生物信息學(xué)預(yù)選定測Abc R 1的三個潛在靶基因BMEII0923,BMEII0372,BMEI1250。它們與布魯氏菌Mer R蛋白家族轉(zhuǎn)錄后調(diào)控、短鏈脫氫酶的活性、周質(zhì)蛋白有關(guān)。通過RACE實驗檢測靶基因的TSS位點,測序驗證后克隆相關(guān)靶基因,然后利用具有綠色熒光GFP報告系統(tǒng)的大腸桿菌質(zhì)粒檢測系統(tǒng),將s RNA構(gòu)建到具有氨芐青霉素抗性的質(zhì)粒載體p ZK001上,將備選targets構(gòu)建到具有氯霉素抗性的質(zhì)粒載體p XG-10SF上,p XG-10SF上具有superfolder GFP基因,可表達(dá)target::GFP融合蛋白。通過共轉(zhuǎn)化兩種質(zhì)粒得到菌落,檢測GFP在特定激發(fā)光下的熒光強度;超聲波破碎菌體后,Western blot檢測GFP蛋白的表達(dá)量;q RT-PCR檢測靶基因的轉(zhuǎn)錄水平,由此推斷GFP蛋白的轉(zhuǎn)錄和表達(dá)情況,從而分析s RNA對于target的調(diào)控作用。相關(guān)結(jié)論如下:1.Target RNA2作為專門的細(xì)菌s RNA靶位點預(yù)測工具,能夠用于布魯氏菌靶位點的分析,例如s RNA Abc R1靶位點的相互作用預(yù)測,但預(yù)測結(jié)果需要更進(jìn)一步的實驗驗證。2.羊種布魯氏菌Brucella menlitensis的s RNA Abc R1經(jīng)驗證是具有調(diào)控功能的非編碼小RNA,能夠下調(diào)靶基因BMEII0923的表達(dá),也能夠上調(diào)靶基BMEII0372的表達(dá),但對于BMEI1250的調(diào)控作用不明顯。3具有GFP的質(zhì)粒報告系統(tǒng)能夠直觀快速的反映羊種布魯氏菌中目的s RNA和潛在靶基因的互作,該報告系統(tǒng)能夠用于布魯氏菌s RNA靶位點的篩選。實驗結(jié)果表明,此大腸桿菌質(zhì)粒報告系統(tǒng)可以用于驗證布魯氏菌s RNA和targets的互作,相比傳統(tǒng)的western blot驗證和q RT-PCR,具有快速直觀的特點。此方法可以便捷的驗證布魯氏菌毒力代謝調(diào)控相關(guān)的s RNA和對應(yīng)靶位點的互作,為揭示布魯氏菌s RNA調(diào)控毒力以及代謝的機理提供新的思路。
[Abstract]:Brucellosis is a gram-negative facultative intracellular parasite that causes brucellosis in humans and livestock and is transmitted through skin, respiratory and digestive tract.Sheep, cattle, pigs and other hosts are infected with abortion and infertility symptoms, while people will develop arthritis, orchitis, wave fever, sweating and other characteristics.After brucella infection, the immune escape mechanism can be established in the organism.Recent studies have shown that there is a class of non-coding small RNA(small non-coding RNAs RNAs in bacteria, which have the potential to regulate bacterial metabolism and virulence related genes.The RNA-seq analysis and prediction of Brucella species showed that s RNA Abc R 1 of Brucella was probably a regulatory function of s RNA Abc R 1.S RNA of Brucella targeted its m RNAs and then played a post-transcriptional regulatory role.Three potential target genes, BMEII0923, BMEII0372 and BMEI1250, were selected by literature homology comparison and bioinformatics to detect Abc R1.They are related to the posttranscriptional regulation of Mer R protein family, the activity of short chain dehydrogenase and the pericytoplasmic protein of Brucella.The TSS site of the target gene was detected by RACE experiment, the relevant target gene was cloned by sequencing, and then the plasmid detection system of E. coli was used to detect the target gene by using the green fluorescent GFP report system.S RNA was constructed into ampicillin resistant plasmid vector p ZK001, and alternative targets was constructed into chloramphenicol resistant plasmid vector p XG-10SF with superfolder GFP gene on p XG-10SF, which could express target::GFP fusion protein.The colony was obtained by co-transformation of two plasmids to detect the fluorescence intensity of GFP under specific excitation light, and the expression of GFP protein was detected by Western blot after supersonic cell fragmentation, and the transcription level of target gene was detected by Q RT-PCR.The transcription and expression of GFP protein were deduced, and the regulation of s RNA on target was analyzed.The relevant conclusions are as follows: 1. Target RNA2, as a special tool for predicting the target sites of bacteria s RNA, can be used for the analysis of target sites of Brucella, such as the interaction prediction of target sites of s RNA Abc R1, but the prediction results need further experimental verification.S RNA Abc R1 of Brucella menlitensis in sheep has been proved to be a non-coding small RNAs with regulatory function, which can down-regulate the expression of target gene BMEII0923 and up-regulate the expression of target BMEII0372.However, the plasmid reporting system with GFP can reflect the interaction of target s RNA and potential target gene in Brucella species directly and quickly. The system can be used to screen target sites of Brucella spp. S RNA.The results showed that the plasmid reporting system could be used to verify the interaction between RNA and targets of Brucella. Compared with the traditional western blot and Q RT-PCR, the system had the characteristics of rapid and intuitive.This method can easily verify the interaction of s RNA and corresponding target sites in the regulation of brucella virulence metabolism, and provide a new idea for revealing the virulence and metabolic mechanism of brucella s RNA.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.614

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1 肖穎;布魯氏菌sRNA AbcR1和靶基因的相互作用研究[D];華中農(nóng)業(yè)大學(xué);2015年

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本文編號：1752535