示踪鼠疫菌构建及吞噬细胞对其吞噬能力的观察

发布时间：2018-06-21 16:05

本文选题：示踪鼠疫菌 + 吞噬作用　；参考：《中国人民解放军军事医学科学院》2011年硕士论文

【摘要】：背景:鼠疫是一种严重威胁人类健康的自然疫源性传染病,主要经蚤叮咬而传播,亦可经呼吸道吸入而感染,传染性强,病死率高。而鼠疫耶尔森氏菌是引起鼠疫的病原体,鼠疫耶尔森氏菌的致病机制与宿主的免疫反应涉及到感染与免疫的两个方面。鼠疫菌通过偶然的机会(蚤类叮咬或人类剥食受感染的动物)进入机体后,首先被局部淋巴组织中的中性粒细胞和巨噬细胞吞噬,被中性粒细胞吞噬的鼠疫菌很快被杀灭,而被巨噬细胞吞噬的鼠疫菌可以在巨噬细胞中大量繁殖,并合成大量的毒力因子。当巨噬细胞崩解,鼠疫菌再次从巨噬细胞中释放到细胞外时,便具备了抵抗宿主免疫防御的能力,然后随淋巴液、血液流经其他局部淋巴组织或全身其他脏器中,引起腺鼠疫,肺鼠疫或败血症鼠疫等。目前对鼠疫菌感染引起宿主体液免疫反应的研究较深入,并发现了一些重要的保护性抗原,如:F1(荚膜抗原)和LcrV(V-抗原)等;然而,越来越多的证据显示细胞免疫在宿主抵抗鼠疫菌的机制中发挥了更加重要的作用,例如,将鼠疫菌活化的T细胞输入小鼠后能有效保护吸入性鼠疫菌的感染,也有证据证实巨噬细胞和中性粒细胞可以有效地吞噬强毒鼠疫菌,但减毒鼠疫菌免疫动物后,巨噬细胞和树突细胞吞噬减毒鼠疫菌的能力和抗原提呈的机制至今尚未阐明。本研究将表达绿色荧光蛋白的原核表达质粒和表达红色荧光蛋白的真核表达质粒导入到鼠疫菌EV76疫苗株和缺失了所有毒力质粒的鼠疫菌201株中,构建成示踪鼠疫菌,拟通过观察巨噬细胞、中性粒细胞和树突细胞对它们的吞噬能力和细胞对鼠疫菌的杀灭能力,为研究宿主免疫反应机制奠定基础。方法:我们将目的片段F1和V以及真核质粒pIRES2 DsRed-Express2经内切酶Bgl II和EcoRI酶切后相连,筛选成功连接,同时目的片段经测序证实无突变的单克隆菌落。提取质粒后,使用鼠疫菌201株质粒缺失株(201P-)和EV76疫苗株作为质粒载体,携带两种类型的质粒(即表达绿色荧光蛋白的原核质粒pBC-GFP和表达红色荧光蛋白的真核质粒pIRES2 DsRed-Express2),其表达不同颜色的荧光蛋白作为鼠疫菌进入机体示踪的手段,利用磁珠分离技术从脾细胞中分离出中性粒细胞、巨噬细胞和树突细胞,利用流式细胞仪检测细胞含荧光蛋白的鼠疫菌比例,来观察三种细胞吞噬鼠疫菌的情况以及三种细胞杀灭鼠疫菌的能力等等。基于本实验主要目的是探究吞噬细胞对鼠疫菌吞噬能力的观察,以及吞噬能力是否与毒力相关等方面,所以我们动物实验只观察了两株菌EV76(pIRES2 DsRed-Express2+pBC-GFP)和201P-(pIRES2 DsRed-Express2-F1 +pBC-GFP),而201P-(pIRES2 DsRed-Express2-V+pBC-GFP)只进行了体外实验,没有再进行动物实验。我们主要通过两株菌携带不同的质粒分别感染两组小鼠来观察吞噬细胞的吞噬情况: EV76(pIRES2 DsRed-Express2+pBC-GFP)和201P-(pIRES2 DsRed-Express2-F1+pBC-GFP),在观察几种细胞吞噬鼠疫菌能力差异的同时,观察田鼠型鼠疫菌201株在质粒完全缺失的情况下作为鼠疫菌DNA疫苗的载体,通过酶联免疫法测定小鼠血清抗体,探讨鼠疫菌201P-作为鼠疫菌DNA疫苗载体的可行性。结果:本实验成功将表达鼠疫菌两个重要保护性抗原的F1和V片段分别克隆到真核表达质粒pIRES2 DsRed-Express2中,经过转染293细胞实验证实插入F1和V片段的真核质粒可以正常表达,将细胞表达的融合蛋白从细胞中释放出来通过SDS-聚丙烯酰胺凝胶电泳及Western-Blot蛋白免疫活性鉴定,结果表明融合蛋白F1和V的免疫原性良好,同时用鼠疫菌感染体外培养的巨噬细胞RAW,分别将细胞内外的鼠疫菌和细胞核染色,通过共聚焦显微镜成功观察巨噬细胞有效地吞噬鼠疫菌,随后用导入两种质粒的鼠疫菌EV76(pIRES2 DsRed-Express2+pBC-GFP)和201P-(pIRES2 DsRed-Express2-F1+pBC-GFP)免疫小鼠,不同时间点处死小鼠后,经过磁珠分离后的巨噬细胞,中性粒细胞,树突细胞经流式细胞仪检测,初步证实24h分离的中性粒细胞中残留有微量的鼠疫菌,而在巨噬细胞和树突细胞内鼠疫菌可以存活,并且流式细胞仪可以检测到表达红色荧光的巨噬细胞和树突细胞。ELISA检测结果显示EV76和201P-(含pIRES2 DsRed-Express2-F1)第一次免疫后均有抗体效价,且相差不大;但第二次加强免疫10天后检测,EV76免疫组抗体滴度明显高于201P-免疫组,而且这两次的检测结果经统计学分析,都具有显著性差异,具有统计学意义。结论:我们设计用两种质粒表达不同的颜色来追踪鼠疫菌进入机体后与吞噬细胞的相互作用,鼠疫菌在存活状态下可以正常表达PBC-GFP原核质粒表达绿色荧光蛋白,使我们可以追踪到鼠疫菌;当鼠疫菌被细胞吞噬并降解掉以后,此时原核质粒在真核环境中不再表达,而真核质粒pIRES2 DsRed-Express2在真核环境中开始表达红色荧光蛋白,有利于我们分析各吞噬细胞吞噬鼠疫菌的能力。本实验将双色质粒导入到鼠疫菌201质粒缺失突变株和EV76疫苗株后,免疫小鼠,对鼠疫菌进入机体后吞噬细胞(包括:中性粒细胞,巨噬细胞和树突细胞)对其吞噬能力以及鼠疫菌的存活情况做了初步评价,在24h中性粒细胞中检测到微量的绿色荧光蛋白,在树突细胞和巨噬细胞中可以检测到相对较多的绿色荧光蛋白,而且EV76株组比201P-组的数量也相对多一些。而且在随后的72h或96h可以检测到表达红色荧光蛋白的巨噬细胞和树突细胞,而且EV组阳性细胞的数量也高于201P-组,初步证明不同吞噬细胞针对鼠疫菌的吞噬能力差异;鼠疫菌的毒力与吞噬细胞的吞噬能力有关;同时我们通过ELISA检测两组小鼠F1抗体的产生情况,通过结果可以看出201P-组同样可以产生F1抗体,虽然两次检测抗体滴度均低于EV76组,但可以证实鼠疫菌201株质粒缺失突变株作为DNA疫苗的载体携带DNA疫苗的可行性。
[Abstract]:Background: plague is a natural infectious disease, which is a serious threat to human health. It is transmitted mainly by the bite of flea. It can also be infected by inhalation of the respiratory tract. It is highly infectious and with high mortality. The plague Jerson Prand is the pathogen of the plague. The mechanism of the Yersinia pestis's disease and the immune response of the host to the host involves infection and immunity. The two aspect. Yersinia pestis can be devoured by neutrophils and macrophages in the local lymphoid tissue, and the Yersinia pestis, phagocytic by neutrophils, can be killed quickly when the Yersinia pestis enters the body by chance (flea bites or human feeding animals), and the pestis phagocytic bacteria phagocytic by macrophage can be large in macrophages. When the macrophages disintegrate and release them from macrophages to the cells again, it has the ability to resist the immune defense of the host, and then with the lymph, the blood flows through other local lymphatic tissues or other organs of the whole body to cause the plague, the plague of the lung, or the plague of septicemia, and so on. A number of important protective antigens, such as F1 (capsule antigen) and LcrV (V- antigen), are found in the study of the host humoral immune response. However, more and more evidence shows that cellular immunity plays a more important role in the mechanism of host resistance to Yersinia pestis, for example, the T cells that are activated by Yersinia pestis are transported. After entering mice, the infection of Yersinia pestis can be effectively protected. There is also evidence that macrophages and neutrophils can effectively phagocytic Yersinia pestis, but the mechanism of phagocytosis and antigen presentation of Yersinia pestis by phagocytic and dendritic cells has not yet been clarified. The prokaryotic expression plasmid of the fluorescent protein and the eukaryotic expression plasmid expressing the red fluorescent protein were introduced into the 201 strains of Yersinia pestis EV76 vaccine strain and the Yersinia pestis missing all the virulence plasmids. The phagocytosis of the macrophages, neutrophils and dendritic cells and the killing of the cells against Yersinia pestis were to be observed. Inability to lay the foundation for studying the host immune response mechanism.
Methods: We screened the target fragment F1 and V and the true nuclear particle pIRES2 DsRed-Express2 through the endonuclease Bgl II and EcoRI enzyme, and screened the successful connection. At the same time, the target fragment was sequenced to confirm the mutagenless colony. After extracting the plasmid, 201 strains of plasmid deletion strain (201P-) and EV76 vaccine strain of Yersinia pestis were used as plasmid carrier, and two were carried. The plasmids of the species (the prokaryotic pBC-GFP expressing green fluorescent protein and the true nuclear particle pIRES2 DsRed-Express2 expressing the red fluorescent protein), which express the different colors of the fluorescent protein as the means to trace the organism as Yersinia pestis, and separate the neutrophils, macrophages and dendrites from the spleen cells by magnetic bead separation technique. Cells, using flow cytometry to detect the proportion of Yersinia pestis in cells containing fluorescent protein, to observe the situation of phagocytosis of Yersinia pestis in three kinds of cells and the ability of three kinds of cells to kill rodent, etc. based on this experiment, the main purpose of this experiment is to explore the phagocytosis of phagocytic phagocytosis and the relationship of phagocytosis to virulence, etc. So we only observed two strains of bacteria EV76 (pIRES2 DsRed-Express2+pBC-GFP) and 201P- (pIRES2 DsRed-Express2-F1 +pBC-GFP), and 201P- (pIRES2 DsRed-Express2-V+pBC-GFP) only carried out in vitro experiments, and no animal experiments were carried out. We mainly infected two groups of mice by two strains of bacteria carrying different plasmids. Phagocytic phagocytosis: EV76 (pIRES2 DsRed-Express2+pBC-GFP) and 201P- (pIRES2 DsRed-Express2-F1+pBC-GFP). At the same time, observing the difference in the ability of several cells to phagocytic Yersinia pestis, the 201 strains of Yersinia pestis were observed as the carrier of the Yersinia pestis DNA vaccine under the condition of complete deletion of the plasmid, and the mouse blood was determined by enzyme immunoassay. To explore the feasibility of Yersinia pestis 201P- as carrier of Yersinia pestis DNA vaccine.
Results: the F1 and V fragments of two important protective antigens of Yersinia pestis were successfully cloned into the eukaryotic expression plasmid pIRES2 DsRed-Express2. Through the transfection of 293 cells, the true nuclear particles inserted into the F1 and V fragments could be expressed normally, and the fusion proteins expressed by the cells were released from the cells through SDS- polypropylene The immunogenicity of the amide gel electrophoresis and Western-Blot protein showed that the immunogenicity of the fusion protein F1 and V was good. At the same time, the infected macrophage RAW was infected with Yersinia pestis in vitro, and the cells were stained with Yersinia pestis and nuclei respectively. The macrophages were successfully observed by confocal microscopy. EV76 (pIRES2 DsRed-Express2+pBC-GFP) and 201P- (pIRES2 DsRed-Express2-F1+pBC-GFP) in mice were immunized with two plasmids. After the mice were killed at different time points, the macrophages, neutrophils and dendritic cells were detected by the flow cytometry after the magnetic beads were separated at different time points. It was preliminarily confirmed that there were trace residues in the neutrophils separated by 24h. Yersinia pestis can survive in the macrophages and dendritic cells, and the flow cytometry can detect the.ELISA detection results of macrophages and dendritic cells expressing red fluorescence. EV76 and 201P- (including pIRES2 DsRed-Express2-F1) have anti body titer after the first immunization, and the difference is small, but the second time of immunization is 10. The titer of the EV76 immunization group was significantly higher than that of the 201P- immune group, and the results of these two tests were statistically significant and statistically significant.
Conclusion: we designed two kinds of plasmids to express different colors to trace the interaction between Yersinia pestis and phagocyte. Yersinia pestis can express the PBC-GFP prokaryotic plasmid to express green fluorescent protein in the condition of survival, so that we can trace Yersinia pestis; when Yersinia pestis is phagocytic and degraded, it is the original. The nucleolus is no longer expressed in the eukaryotic environment, and the true nucleolus pIRES2 DsRed-Express2 begins to express the red fluorescent protein in the eukaryotic environment, which is beneficial to our analysis of the phagocytic phagocytosis of the phagocytosis. This experiment introduced the double color plasmid into the 201 plasmid missing mutant strain and the EV76 vaccine strain of Yersinia pestis, and immunized mice and Yersinia pestis. The phagocytic cells (including neutrophils, macrophages and dendritic cells) were evaluated for their phagocytosis and the survival of Yersinia pestis after entering the body. A small amount of green fluorescent protein was detected in 24h neutrophils, and relatively more green fluorescent protein could be detected in dendritic cells and macrophages, and EV76 The number of the plant group was also more than that of the 201P- group. Moreover, the macrophages and dendritic cells expressing the red fluorescent protein were detected in the subsequent 72h or 96h, and the number of the positive cells in the group EV was also higher than that in the 201P- group. At the same time, we detected the production of F1 antibody in two groups of mice by ELISA. Through the results, we can see that the 201P- group could also produce F1 antibody, although the titer of the two test antibody was lower than that of the EV76 group, but it could be proved that the 201 strains of the plasmid missing mutant strain of Yersinia pestis were feasible to carry the DNA vaccine as a carrier of DNA vaccine.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

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