钩端螺旋体Lig蛋白在仓鼠动物模型中的免疫效果评价

发布时间：2018-07-26 13:51

【摘要】：钩端螺旋体病是一种全球分布重要的急性人兽共患病，由钩端螺旋体属致病性问号钩端螺旋体引起，在世界范围内广泛流行，对人类的健康和畜牧业经济发展产生了严重的威胁。从事户外职业，洪水暴发，与动物密切接触等因素，会增加此病的传播风险，加大感染机率。钩端螺旋体病有多种临床症状，包括亚临床，自限，无黄疸，发热等，严重程度可到致死症状。钩端螺旋体广泛分布在热带，亚热带和温带的城市及农村地区。在过去的几十年中，流行病学模式的变化强调了该病作为一个公共健康问题的重要性。每年报道的钩体病例有50万余例，超过10%的病死率。我国受钩体病疫情困扰已久，令其防治成为一项迫在眉睫的任务，但已取得的研究进展中对致病机制和宿主免疫反应机理的研究还未十分清楚，普遍认为钩端螺旋体的感染过程主要是在各种膜表面分子（毒力因子）的帮助下侵入和粘附特定宿主细胞，形成持久性定殖和免疫逃避。由于钩端螺旋体LPS碳水化合物的组成有差异性，使得钩体菌型复杂，血清型多样，超过280种。LPS作为钩端螺旋体的一种免疫保护性抗原，已经被用于研制疫苗，但商品化的灭活全菌苗或弱毒苗会产生一些毒性反应，需要加强免疫来维持免疫时间，同时不同血清型的LPS成分不同，对其他血清型无法产生交叉保护作用。现有研究表明，，致病性钩体的一些外膜蛋白（OMP）如OmpL1、LipL32、LipL41、LigA、LigB、Lp1454等在各种血清型之间存在抗原保守性，因此免疫后可以对多种血清型起到交叉保护作用，作为候选亚单位疫苗表现出良好的发展前景，有可能替代全细胞灭活疫苗。钩端螺旋体通过表面粘附素作用结合到宿主细胞外基质（ECM）引发感染。在钩体外表面暴露的多种粘附素中，Lig蛋白含量丰富且较为保守，广泛分布在各种致病性血清型中。本试验选择问号钩端螺旋体秋季型临4株基因组为模板，目标基因为LigAcon、LigAvar、LigBcen1，采用高保真PCR扩增片段测序后，分别与原核表达载体pGEX-4T-1连接构建重组表达载体pGEX-LigAcon，pGEX-LigAvar、pGEX-LigBcen1转入大肠杆菌BL21(DE3)中诱导表达重组蛋白，将亲和层析纯化后的可溶性蛋白混合弗氏佐剂，作为免疫抗原皮下注射3~4周龄的仓鼠，试验组为rLigAcon、rLigAvar、rLigBcen1、rLigAcon+rLigAvar，3周后加强免疫，对照组PBS组和GST标签组相同免疫程序。重组蛋白的免疫保护效果由仓鼠模型中存活率和组织病理学切片观察来评价。试验结果显示诱导表达的融合蛋白大小分别约为89kDa(LigAcon)、92kDa(LigAvar)、68kDa(LigBcen1)，试验组中免疫的重组蛋白LigA对急性感染钩体的仓鼠有明显保护作用，与PBS对照组、GST标签组对比，差异显著（P＜0.05）。病理学切片显示存活的仓鼠肝、肾、肺脏组织内的炎症反应和出血状况与对照组相比明显改善。本研究中体现了Lig蛋白作为问号钩端螺旋体属的特异性保护抗原的免疫保护效果，钩端螺旋体LigA基因对抵抗同型钩体感染能力较好，这些初步试验结果为钩端螺旋体亚单位疫苗的进一步研究奠定了基础。
[Abstract]:Leptospirosis, an important acute zoonosis in the world, is caused by the leptospirosis, which is caused by the leptospirosis, which is widely popular in the world, and poses a serious threat to the health of human beings and the development of animal husbandry. The factors such as outdoor occupation, flood outbreaks, close contact with animals, and so on, will increase Leptospirosis has a variety of clinical symptoms, including subclinical, self limiting, non jaundice, fever, and fatal symptoms. Leptospirosis is widely distributed in tropical, subtropical and temperate cities and rural areas. In the past few decades, changes in epidemiological patterns have emphasized this The importance of disease as a public health problem is reported. There are more than 500 thousand cases of leptospirosis reported annually, with a mortality rate of more than 10%.
The prevention and control of leptospirosis has become an urgent task in China for a long time. However, the research progress on the pathogenesis and host immune response mechanism is not very clear. It is generally believed that the infection process of leptospirosis is mainly under the help of various membrane surface molecules (virulence factors) to invade and adhere. Specific host cells, forming persistent colonization and immune escape. Due to the difference in the composition of carbohydrates of the Leptospira LPS, the Leptospira type is complex and the serotypes are diverse, more than 280.LPS as an immuno protective antigen of the Leptospira, which has been used in the study of vaccines, but commercially inactivated total or weakly poisonous seedlings will be inactivated. To produce some toxic reactions, we need to strengthen the immune system to maintain the immune time. At the same time, the LPS components of different serotypes are different, and there is no cross protection to other serotypes.
Existing studies have shown that some of the outer membrane proteins (OMP) of pathogenic Leptospira such as OmpL1, LipL32, LipL41, LigA, LigB, Lp1454, and so on are conserved in various serotypes. Therefore, the immunization can play a cross protective effect on a variety of serotypes. As a candidate subunit vaccine, it has a good prospect of development and may replace whole cell death. Live vaccines.
The leptospirosis is associated with the extracellular matrix (ECM) of the host by adhesion to the host cell matrix (ECM). In a variety of adhesion elements exposed to the surface of the hook, the content of Lig protein is rich and conservative, widely distributed in various pathogenicity serotypes. This test selected the genome of the 4 strains of leptospirosis in the Leptospira as a template and the target gene. For LigAcon, LigAvar, LigBcen1, the recombinant expression vector pGEX-LigAcon, pGEX-LigAvar, pGEX-LigBcen1 was transferred into Escherichia coli BL21 (DE3) to express the recombinant protein after sequencing with high fidelity PCR amplification fragment, and pGEX-LigAvar, pGEX-LigBcen1 was transferred into Escherichia coli BL21 (DE3), and the soluble protein mixed with purified soluble protein after affinity chromatography was used as a free adjuvant. The pestilence antigen was injected subcutaneously for 3~4 weeks old hamster. The test group was rLigAcon, rLigAvar, rLigBcen1, rLigAcon+rLigAvar. After 3 weeks, the immunization was strengthened. The immune protection of the control group was the same as that of the PBS group and the GST label group. The immune protective effect of the recombinant protein was evaluated by the survival rate and histopathology section of the hamster model.
The results showed that the size of the induced fusion protein was about 89kDa (LigAcon), 92kDa (LigAvar), 68kDa (LigBcen1). The recombinant protein LigA in the test group had obvious protective effect on the hamster with acute infection of the leptospira. Compared with the PBS control group, the GST label group was significantly different (P < 0.05). The pathological section showed the surviving hamster liver and kidney. The inflammatory response and bleeding status in the lung tissue were significantly improved compared with those in the control group. In this study, the Lig protein was used as a specific protective antigen of the Leptospira interroponate, and the Leptospira LigA gene was better in resistance to the same type of Leptospira. These preliminary results were the Leptospira subunit. Further research has laid the foundation for the vaccine.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855

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