问号钩端螺旋体属特异性外膜蛋白LipL41抗原表位预测及其有效表位噬菌体展示肽的筛选

发布时间：2018-07-09 11:36

本文选题：问号钩端螺旋体 + 属特异性外膜蛋白　；参考：《浙江大学》2008年硕士论文

【摘要】： 背景和目的钩端螺旋体(Leptospirosis)(简称钩体)病是由问号钩体(Leptospira interrogans)感染引起的自然疫源性人兽共患传染病,世界范围内广泛流行,尤其多见于水稻种植区,我国也是该病流行的主要疫区之一。问号钩体自然宿主众多,多可在其肾脏长期生存繁殖,并可随尿液排出,污染土壤和水源,人类常可通过接触疫土和疫水而感染,因而钩体病也是各国洪涝灾害时重点防疫和监控的传染病之一。接种疫苗是预防和控制钩体病的最有效措施,但钩体血清群、型众多,各血清群、型抗体之间交叉保护作用较弱或无。不同国家甚至同一国家不同地区主要流行的问号钩体血清群、型差异很大,常随当地自然宿主变更而改变,给钩体病预防带来很大困难。目前普遍使用的钩体疫苗是根据当地流行的数种优势钩体血清群、型制备的多价钩体全菌死疫苗,交叉保护作用弱且副作用较大。多价钩体外膜疫苗是由我国科学家首先研制成功的组分疫苗,虽显示免疫效果好和副作用小等优点,但交叉保护作用较差的缺陷仍未得到改变。位于钩体外膜上的外膜蛋白(Outer membrane proteins,OMPs)在决定抗原特异性方面有重要作用,其中一些蛋白为钩体属特异性抗原,例如LipL41,它们的编码基因几乎保守的存在于所有致病钩体中,在问号钩体基因工程疫苗的研制上有广泛的前景。外膜脂蛋白LipL41由355个氨基酸组成,在钩体的内外两层膜中都有分布,并暴露于外膜表面。Haake等(2000)克隆出lipL41全长基因序列,并发现它只存在于致病钩体中,而在非致病钩体中则测不到。我们以往的研究结果也证实我国15群15型问号钩体标准参考株均有lipL41基因,但存在两个不同的基因型lipL41/1和lipL41/2,其中lipL41/1基因型包含了我国人群中流行最广的问号钩体黄疸出血群、波摩那群、流感伤寒群、秋季群和赛罗群等,而lipL41/2基因型所包含的钩体血清群在国内的发病率都较低。此外研究还发现多数问号钩体临床分离株都含有ipL41基因,并且表达LipL41蛋白,在多数钩体病人的血清中也可测到相应保护性抗体的存在。含多个和多种抗原表位(epitope)的多抗原肽(multiple antigenicpeptide,MAP)是近年发展起来的一种研制新型基因工程疫苗的先进技术和研究策略,是解决普通钩体疫苗交叉保护作用弱的有效途径,而鉴定有效的抗原表位是研制MAP的第一步。研究抗原表位不仅可深层次的揭示蛋白抗原分子诱导免疫应答的分子机制及特点,而且在研制新型分子疫苗方面也有重要应用前景。通过噬菌体展示系统对LipL41/1和LipL41/2的T和B有效联合表位进行鉴定,可为进一步研制钩体MAP疫苗奠定基础;序列对比发现两个基因型存在少量氨基酸序列差异,且某些残基的差异使两个基因型的某些预测表位产生了较为显著的差异,对这些有差异的表位进行鉴定有利于深入了解其抗原性和免疫反应性特点及个别氨基酸序列突变对其表位抗原性的影响,同时也可为下一步评估MAP疫苗的保护范围提供参考。实验方法采用生物信息学技术对LipL41/1和LipL41/2的T细胞和B细胞表位进行预测和分析。选择主要T细胞和B细胞联合表位肽并融合表达于M13KE噬菌体PⅢ蛋白上,PEG/NaCl沉淀法提纯重组噬菌体,SDS-PAGE鉴定目的重组PⅢ蛋白(rPⅢ)。分别以自制备rLipL41/1、rLipL41/2、黄疸出血群赖型赖株血清和病人抗血清为一抗,采用Western blot对上述表位肽进行鉴定和筛选。结果通过抗原表位预测,选择了6个LipL41/1和2个LipL41/2联合表位。经扩增获得了预期的各抗原表位片段。各目的表位序列均准确插入噬菌体PⅢ蛋白N端并有效表达。各抗血清均能识别上述8个联合表位,但杂交信号强度存在差异,表现在两个方面:一是用同一抗血清为一抗时,不同表位的杂交信号强度不同,另一方面是同一表位对不同抗血清的相对信号强度也存在差异。表位中或附近的氨基酸序列突变可能会对表位的抗原性产生一定的影响,既可以使突变后表位与原抗体的亲和力升高也可表现为降低。综合8个表位对不同抗血清的Western Blot结果及实际意义,表位LipL41/1-30和LipL41/1-233的信号强度在兔抗全菌血清和多数病人血清中的杂交信号强度均较强,其余表位的信号强度较弱或变异较大。结论本研究成功构建了T细胞和B细胞联合表位肽段的噬菌体展示系统,所采用的生物信息学技术可有效的预测抗原表位。实验鉴定所构建的8个表位均为LipL41的有效抗原表位,其中表位LipL41/1-30和LipL41/1-233可应用于钩体MAP疫苗研制。
[Abstract]:Background and objective Leptospira (Leptospirosis) disease is a natural zoonotic infectious disease caused by Leptospira interrogans infection. It is widely prevalent in the world, especially in rice planting area. China is also one of the main epidemic areas in this disease. In the long-term survival of the kidney, and can be excreted with urine, pollution of soil and water, human can often be infected by contact with plague soil and epidemic water, so leptospirosis is also one of the infectious diseases which are the key epidemic prevention and control in the flood and waterlogging disasters in various countries.
The immunization is the most effective measure to prevent and control Leptospira disease, but the Leptospira serogroup has a large number, and the cross protection between the serum groups and the types of antibodies is weak or not. The serogroups of the interropid in different countries and even in different regions of the same country vary greatly, often changing with the local natural hosts and preventing leptospiriosis. The widely used Leptospira vaccine is based on the local epidemic of several dominant Leptospira serogroups, the polyvalent whole bacteria dead vaccine prepared by the type of Leptospira, the cross protective effect is weak and the side effects are large. The multivalent Leptospira epicardial vaccine is the first successful component vaccine developed by Chinese scientists, although it shows good immune effect and side effects. Outer membrane proteins (OMPs), which is located in the outer membrane of the Leptospira, plays an important role in determining antigen specificity, some of which are specific antigens of the Leptospira, such as LipL41, and their coding genes are almost conservative in all pathogenic hooks. It has broad prospects in the development of genetic engineering vaccine for Leptospira interrogation.
The outer membrane lipoprotein LipL41 is composed of 355 amino acids and is distributed in the outer and outer two layers of the leptospira. And exposed to the outer membrane of the outer membrane,.Haake and so on (2000) cloned the lipL41 full length gene sequence, and found that it exists only in the pathogenic Leptospira, but not in the non pathogenic Leptospira. Our previous research results also confirmed the 15 groups of the 15 questions in our country. The standard reference strain of the Leptospira has lipL41 gene, but there are two different genotypes lipL41/1 and lipL41/2, in which the lipL41/1 genotype contains the most popular leptospirosis jaundice haemorrhage group, the bomonna group, the influenza typhus group, the autumn group and the serogroup, and the lipL41/2 genotypes contain the leptospirosis serogroup in China. The rate of disease is low. In addition, most of the clinical isolates of the Leptospira contain ipL41 gene, and the expression of LipL41 protein can also be detected in the serum of most leptospirosis patients.
Multiple antigenicpeptide (MAP), which contains multiple and multiple antigen epitopes (epitope), is an advanced technology and research strategy developed in recent years to develop a new genetic engineering vaccine. It is an effective way to solve the weak cross protection of common Leptospira vaccine. Identification of effective antigen epitopes is the first step in the development of MAP. The antigen epitopes not only reveal the molecular mechanism and characteristics of the immune response induced by protein antigen molecules in a deep level, but also have an important application prospect in the development of new molecular vaccines. Through the phage display system, the effective joint epitopes of T and B of LipL41/1 and LipL41/2 can be established for the further development of the Leptospira MAP vaccine. The sequence comparison found that there was a small number of amino acid sequences in the two genotypes, and the differences in some residues made some significant differences in some prediction epitopes of the two genotypes. Identification of these different epitopes was helpful to understand their antigenicity and immunoreactivity characteristics and the mutation of individual amino acid sequences. The antigenicity of epitopes may also provide a reference for further evaluation of the protective range of MAP vaccine.
The experimental methods used bioinformatics to predict and analyze the epitopes of T and B cells of LipL41/1 and LipL41/2. Select the joint epitope of the main T cells and B cells and fused on the M13KE phage P III protein. The recombinant phage was purified by PEG/NaCl precipitation. SDS-PAGE was determined to restructure P III protein (rP III). RLipL41/1, rLipL41/2, jaundice haemorrhagic group Lai strain and anti sera from patients were taken as one antibody. Western blot was used to identify and screen the above epitope peptides.
Results 6 LipL41/1 and 2 LipL41/2 combined epitopes were selected through the epitope prediction. The expected epitopes were amplified by amplification. Each epitope sequence was accurately inserted into the N terminal of the phage P III protein and expressed effectively. All the antiserum could identify the above 8 joint epitopes, but the signal intensity of the hybridization was different, showing two. One is: first, when the same antiserum is used as one resistance, the intensity of hybrid signals in different epitopes is different, on the other hand, the relative signal intensity of different antisera is also different from the same epitopes. The mutation of amino acid sequence in the epitopes or near the epitopes may have a certain effect on the antigenicity of the epitopes, which can make the mutation epitopes and the original resistance. Western Blot results and practical significance of 8 epitopes to different antiserum, the signal intensity of epitopes LipL41/1-30 and LipL41/1-233 were stronger in Rabbit anti whole sera and in most of the patients' sera, and the signal intensity of the other epitopes was weaker or larger than that of the other epitopes.
Conclusion the study successfully constructed the phage display system of the joint epitope of T cells and B cells. The bioinformatics techniques used can effectively predict the epitopes of the antigen. The 8 epitopes constructed by the experimental identification are all effective antigen epitopes of LipL41, and the epitope LipL41/1-30 and LipL41/1-233 can be applied to the development of the MAP vaccine of the leptospira.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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