炭疽杆菌S-层基因sap和eag的功能研究

发布时间：2018-06-26 10:22

本文选题：炭疽杆菌 + Cre/loxp系统　；参考：《华中农业大学》2011年硕士论文

【摘要】：炭疽芽孢杆菌(Bacillus anthracis)简称炭疽杆菌,是能形成芽孢的革兰氏阳性杆菌；它可感染动物和人引起皮肤炭疽、肺炭疽和肠炭疽等。炭疽是五大人畜共患病之一,对畜牧业及人类健康的危害很大。在炭疽杆菌的毒力调控网络中除已知质粒pXO1和pXO2上的毒力基因外,在染色体上还存在一些与毒力相关的因子,曾有研究报道炭疽杆菌的S-层蛋白也可能是毒力因子。炭疽杆菌的S-层蛋白主要为表面排列蛋白(Surface array protein, Sap)和胞外抗原1(Extracellular antigen 1, EA1),分别由sap和eag基因编码。为了研究炭疽杆菌S-层蛋白功能,本研究首先利用Cre/loxp系统在同源重组的原理下构建不带任何抗性标记的目的基因sap和eag的缺失突变株,在核酸水平及蛋白质水平上进行验证；然后,通过测定生长曲线、糖代谢及电镜观察菌体表面形态来比较说明缺失基因后是否影响炭疽杆菌A16R的生理生化表型：通过不同的动物模型评价A16R与其突变株的毒力差异；利用双向电泳技术比较A16R与突变株间的差异蛋白,通过质谱鉴定差异蛋白,分析缺失基因后对A16R其他蛋白的影响。主要结果如下： 1无抗性A16R△sap和双缺失突变株A16R△sap△eag的构建为了研究sap及eag基因的功能,敲除目的基因构建缺失突变株是研究的基础,本研究无痕(不带抗性标记)敲除的方法为后期疫苗的应用提供更广阔的空间。以炭疽杆菌疫苗株A16R为出发菌株,利用pKSV7穿梭质粒构建打靶载体,将其转入A16R中,同源重组筛选突变株A16R△sap::spc,再利用Cre/loxp系统在同源重组的原理下去除抗性基因,获得无痕sap突变株A16R△sap;同样的方法构建了双缺失△sap△eag突变株A16R△sap△eag。 2 sap或eag基因单/双缺失株生理生化和S-层结构的分析为了分析sap和eag基因对A16R的生理生化或表型的影响,比较A16R及其突变株生长曲线、49种糖代谢情况发现,sap或eag基因单/双缺失后对A16R的生理生化方面没有影响,而通过透射电镜观察菌体S-层结构发现在双缺失sap和eag基因后A16R不形成S-层,两个单缺失株形成的S-层结构分别比A16R薄7nm或19nm。 3应用动物模型对A16 R与其突变株的毒力进行评价为了验证sap和eag基因是否为毒力相关基因,通过动物模型的攻毒实验能直观的体现出缺失基因后的毒力变化,A16R及其突变株的芽孢和繁殖体攻毒不同动物实验结果均说明炭疽杆菌S-层基因eag缺失使A16R毒力明显减弱,而sap基因缺失后对A16R毒力没有影响。 4 A16R与其突变株的比较蛋白质组学研究制备A16R及其突变株各个时相的全菌体蛋白样品,完成其pH4-7梯度的双向电泳图谱,比较双向图谱中的差异蛋白点,共获得207个差异点,缺失S-层基因sap或eag后主要影响的功能蛋白为能量合成和转换相关的蛋白；通过比较蛋白质组学鉴定到的差异蛋白中未发现其他与毒力相关的蛋白。质谱鉴定到炭疽杆菌假想S-层蛋白BA3338；当缺失sap基因后,BA3338表达量在对数期时明显上调,当缺失eag基因后,对数末期时BA3338的表达量一个上调一个下调,而稳定期及衰亡期其表达均上调；因Sap和EA1表达具有时相性,即对数期时主要表达的S-层蛋白为Sap,进入稳定期后EA1为主要的S-层蛋白；说明BA3338随着时相的改变其蛋白功能互补了Sap和EA1的蛋白功能。双缺失菌株未鉴定到BA3338的表达变化。 A16R与A16R△asp△eag双向电泳图谱比较发现在双缺失Sap和EA1蛋白后有两个关于芽孢形成的蛋白(Ad07,Ad18)发生了缺失,而在培养双缺失株的芽孢时并未见其形成芽孢的能力发生改变；说明在A16R中这两个蛋白的缺失并不影响芽孢的形成。动物攻毒实验和比较蛋白质组学分析A16R及其突变株的差异蛋白结果均可以认定eag是炭疽杆菌的一个毒力相关因子,而sap仅作为结构基因表达Sap蛋白构成炭疽杆菌的S-层结构。本研究不仅为炭疽杆菌S-层基因功能研究提供依据,更重要的是促进安全有效的炭疽杆菌疫苗的研制,并有助于完善治疗方法。
[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.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378.72

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