本文選題：炭疽桿菌 + Cre/loxp系統(tǒng)��； 參考：《華中農(nóng)業(yè)大學(xué)》2011年碩士論文

【摘要】：炭疽芽孢桿菌(Bacillus anthracis)簡(jiǎn)稱炭疽桿菌,是能形成芽孢的革蘭氏陽(yáng)性桿菌；它可感染動(dòng)物和人引起皮膚炭疽、肺炭疽和腸炭疽等。炭疽是五大人畜共患病之一,對(duì)畜牧業(yè)及人類健康的危害很大。在炭疽桿菌的毒力調(diào)控網(wǎng)絡(luò)中除已知質(zhì)粒pXO1和pXO2上的毒力基因外,在染色體上還存在一些與毒力相關(guān)的因子,曾有研究報(bào)道炭疽桿菌的S-層蛋白也可能是毒力因子。炭疽桿菌的S-層蛋白主要為表面排列蛋白(Surface array protein, Sap)和胞外抗原1(Extracellular antigen 1, EA1),分別由sap和eag基因編碼。 為了研究炭疽桿菌S-層蛋白功能,本研究首先利用Cre/loxp系統(tǒng)在同源重組的原理下構(gòu)建不帶任何抗性標(biāo)記的目的基因sap和eag的缺失突變株,在核酸水平及蛋白質(zhì)水平上進(jìn)行驗(yàn)證；然后,通過(guò)測(cè)定生長(zhǎng)曲線、糖代謝及電鏡觀察菌體表面形態(tài)來(lái)比較說(shuō)明缺失基因后是否影響炭疽桿菌A16R的生理生化表型：通過(guò)不同的動(dòng)物模型評(píng)價(jià)A16R與其突變株的毒力差異；利用雙向電泳技術(shù)比較A16R與突變株間的差異蛋白,通過(guò)質(zhì)譜鑒定差異蛋白,分析缺失基因后對(duì)A16R其他蛋白的影響。主要結(jié)果如下： 1無(wú)抗性A16R△sap和雙缺失突變株A16R△sap△eag的構(gòu)建 為了研究sap及eag基因的功能,敲除目的基因構(gòu)建缺失突變株是研究的基礎(chǔ),本研究無(wú)痕(不帶抗性標(biāo)記)敲除的方法為后期疫苗的應(yīng)用提供更廣闊的空間。以炭疽桿菌疫苗株A16R為出發(fā)菌株,利用pKSV7穿梭質(zhì)粒構(gòu)建打靶載體,將其轉(zhuǎn)入A16R中,同源重組篩選突變株A16R△sap::spc,再利用Cre/loxp系統(tǒng)在同源重組的原理下去除抗性基因,獲得無(wú)痕sap突變株A16R△sap;同樣的方法構(gòu)建了雙缺失△sap△eag突變株A16R△sap△eag。 2 sap或eag基因單/雙缺失株生理生化和S-層結(jié)構(gòu)的分析 為了分析sap和eag基因?qū)16R的生理生化或表型的影響,比較A16R及其突變株生長(zhǎng)曲線、49種糖代謝情況發(fā)現(xiàn),sap或eag基因單/雙缺失后對(duì)A16R的生理生化方面沒(méi)有影響,而通過(guò)透射電鏡觀察菌體S-層結(jié)構(gòu)發(fā)現(xiàn)在雙缺失sap和eag基因后A16R不形成S-層,兩個(gè)單缺失株形成的S-層結(jié)構(gòu)分別比A16R薄7nm或19nm。 3應(yīng)用動(dòng)物模型對(duì)A16 R與其突變株的毒力進(jìn)行評(píng)價(jià) 為了驗(yàn)證sap和eag基因是否為毒力相關(guān)基因,通過(guò)動(dòng)物模型的攻毒實(shí)驗(yàn)?zāi)苤庇^的體現(xiàn)出缺失基因后的毒力變化,A16R及其突變株的芽孢和繁殖體攻毒不同動(dòng)物實(shí)驗(yàn)結(jié)果均說(shuō)明炭疽桿菌S-層基因eag缺失使A16R毒力明顯減弱,而sap基因缺失后對(duì)A16R毒力沒(méi)有影響。 4 A16R與其突變株的比較蛋白質(zhì)組學(xué)研究 制備A16R及其突變株各個(gè)時(shí)相的全菌體蛋白樣品,完成其pH4-7梯度的雙向電泳圖譜,比較雙向圖譜中的差異蛋白點(diǎn),共獲得207個(gè)差異點(diǎn),缺失S-層基因sap或eag后主要影響的功能蛋白為能量合成和轉(zhuǎn)換相關(guān)的蛋白；通過(guò)比較蛋白質(zhì)組學(xué)鑒定到的差異蛋白中未發(fā)現(xiàn)其他與毒力相關(guān)的蛋白。 質(zhì)譜鑒定到炭疽桿菌假想S-層蛋白BA3338；當(dāng)缺失sap基因后,BA3338表達(dá)量在對(duì)數(shù)期時(shí)明顯上調(diào),當(dāng)缺失eag基因后,對(duì)數(shù)末期時(shí)BA3338的表達(dá)量一個(gè)上調(diào)一個(gè)下調(diào),而穩(wěn)定期及衰亡期其表達(dá)均上調(diào)；因Sap和EA1表達(dá)具有時(shí)相性,即對(duì)數(shù)期時(shí)主要表達(dá)的S-層蛋白為Sap,進(jìn)入穩(wěn)定期后EA1為主要的S-層蛋白；說(shuō)明BA3338隨著時(shí)相的改變其蛋白功能互補(bǔ)了Sap和EA1的蛋白功能。雙缺失菌株未鑒定到BA3338的表達(dá)變化。 A16R與A16R△asp△eag雙向電泳圖譜比較發(fā)現(xiàn)在雙缺失Sap和EA1蛋白后有兩個(gè)關(guān)于芽孢形成的蛋白(Ad07,Ad18)發(fā)生了缺失,而在培養(yǎng)雙缺失株的芽孢時(shí)并未見(jiàn)其形成芽孢的能力發(fā)生改變；說(shuō)明在A16R中這兩個(gè)蛋白的缺失并不影響芽孢的形成。 動(dòng)物攻毒實(shí)驗(yàn)和比較蛋白質(zhì)組學(xué)分析A16R及其突變株的差異蛋白結(jié)果均可以認(rèn)定eag是炭疽桿菌的一個(gè)毒力相關(guān)因子,而sap僅作為結(jié)構(gòu)基因表達(dá)Sap蛋白構(gòu)成炭疽桿菌的S-層結(jié)構(gòu)。 本研究不僅為炭疽桿菌S-層基因功能研究提供依據(jù),更重要的是促進(jìn)安全有效的炭疽桿菌疫苗的研制,并有助于完善治療方法。
[Abstract]:Bacillus anthracis (Bacillus anthracis), abbreviated as Bacillus anthracis, is a gram positive bacillus that forms spores; it can infect animals and people to cause skin anthrax, pulmonary anthrax and intestinal anthrax. Anthrax is one of the five adult zoonosis, which is very harmful to animal husbandry and human health. It is known in the virulence control network of Bacillus anthracis. Besides the virulence genes on the plasmid pXO1 and pXO2, there are some virulence related factors on the chromosome. The S- layer protein of Bacillus anthracis has been reported to be also a virulence factor. The S- layer protein of Bacillus anthracis is mainly the surface permutation protein (Surface array protein, Sap) and the extracellular antigen 1 (Extracellular antigen 1, EA1), respectively. Encoded by SAP and EAG genes.
In order to study the function of S- layer protein of Bacillus anthracis, this study first constructed the deletion mutant of the target gene sap and EAG without any resistance marker under the principle of homologous recombination by using the Cre/loxp system to verify the nucleic acid level and protein level. Then, the growth curve, sugar metabolism and electron microscopy were used to observe the surface shape of the mycelium. Whether the deletion gene affects the physiological and biochemical phenotype of the Bacillus anthracis A16R: evaluate the difference in virulence between A16R and its mutant by different animal models; compare the difference proteins between the A16R and the mutant strain by two dimensional electrophoresis, identify the difference protein by mass spectrometry, and analyze the shadow of the other A16R protein after the deletion gene. The main results are as follows:
Construction of non resistant A16R delta sap and double deletion mutant A16R delta sap sap EAG
In order to study the function of sap and EAG genes, knockout the deletion mutant of the target gene is the basis of the study. This study provides a wider space for the application of the later vaccine. The vaccine strain A16R of Bacillus anthracis vaccine strain is used as the starting strain, and the target vector is constructed by the pKSV7 shuttle plasmid and transferred into the A16R. The homologous recombination screening mutant A16R Delta sap:: SPC, and then using the Cre/loxp system in the homologous recombination principle to remove the resistant gene and obtain the sap mutant A16R delta sap; the same method constructs the dual deletion delta SAP delta EAG mutant A16R delta SAP Delta eag..
Analysis of physiological and biochemical and S- layer structure of 2 SAP / EAG single / double deletion strains
In order to analyze the effects of sap and EAG genes on the physiological, biochemical or phenotypic effects of A16R, the growth curves of A16R and its mutant strains were compared. The 49 kinds of glucose metabolism found that the single / double deletion of sap or EAG genes had no effect on the physiological and biochemical aspects of A16R, but it was observed by transmission electron microscopy that the present double deletion sap and EAG genes were not formed. Layer, the S- layer structure formed by two single deletion strains is thinner than that of A16R or 7Nm 19nm.
3 animal models were used to evaluate the virulence of A16 R and its mutants.
In order to verify whether the sap and EAG genes are virulence related genes, the toxicity of the missing genes can be visualized through the attack experiments of the animal model. The results of the A16R and the spores of the mutant and the experimental results of the different animals in the breeding body indicate that the S- layer of the Bacillus anthracis S- gene EAG deletion makes the A16R virulence significantly weakened, and the sap gene is missing after the deletion of the gene. There is no effect on the virulence of A16R.
Comparative proteomics study of 4 A16R and its mutants
The whole bacterial protein samples of each phase of A16R and its mutant were prepared, and the two-dimensional electrophoresis Atlas of its pH4-7 gradient was completed, and the difference protein points in the bi-directional map were compared, and 207 differences were obtained. The functional proteins which were mainly affected by the deletion of the S- layer gene sap or EAG were the energy synthesis and transfer related proteins. No other virulence related proteins were found in the differentially expressed proteins.
The mass spectrum identified the hypothetical S- layer protein BA3338 of Bacillus anthracis; when the sap gene was missing, the expression of BA3338 was obviously up-regulated at logarithmic phase. After the deletion of the EAG gene, the expression of BA3338 was up regulated by a down-regulation at the end of logarithmic stage, and the expression of the expression was up up in the stable period and in the decay period. The expression of Sap and EA1 was of the temporal phase, that is the logarithmic phase. The expression of S- layer protein is Sap, and EA1 is the main S- layer protein after entering the stable period. It shows that the function of Sap and EA1 complements the protein function of BA3338 with the change of the time phase. The double deletion strain has not identified the change of BA3338 expression.
A16R and A16R delta ASP delta EAG bi-directional electrophoresis atlas found that there were two proteins (Ad07, Ad18) formed after double deletion of Sap and EA1 protein (Ad07, Ad18), but the ability to form spore was not changed when the spores were cultured with double missing strains, indicating that the absence of the two proteins in A16R did not affect the formation of spores.
Animal toxicity test and comparative proteomics analysis of A16R and the differential protein results of the mutant strain can be found that EAG is a virulence related factor of Bacillus anthracis, and SAP only acts as the structural gene expression Sap protein to form the S- layer structure of Bacillus anthracis.
This study not only provides the basis for the study of the gene function of the S- layer of Bacillus anthracis, but also is important to promote the development of the safe and effective Bacillus anthracis vaccine and to improve the treatment method.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R378.72

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