本文選題：問號(hào)鉤端螺旋體 + 屬特異性外膜蛋白��； 參考：《浙江大學(xué)》2008年碩士論文

【摘要】： 背景和目的鉤端螺旋體(Leptospirosis)(簡(jiǎn)稱鉤體)病是由問號(hào)鉤體(Leptospira interrogans)感染引起的自然疫源性人獸共患傳染病,世界范圍內(nèi)廣泛流行,尤其多見于水稻種植區(qū),我國也是該病流行的主要疫區(qū)之一。問號(hào)鉤體自然宿主眾多,多可在其腎臟長(zhǎng)期生存繁殖,并可隨尿液排出,污染土壤和水源,人類�？赏ㄟ^接觸疫土和疫水而感染,因而鉤體病也是各國洪澇災(zāi)害時(shí)重點(diǎn)防疫和監(jiān)控的傳染病之一。 接種疫苗是預(yù)防和控制鉤體病的最有效措施,但鉤體血清群、型眾多,各血清群、型抗體之間交叉保護(hù)作用較弱或無。不同國家甚至同一國家不同地區(qū)主要流行的問號(hào)鉤體血清群、型差異很大,常隨當(dāng)?shù)刈匀凰拗髯兏淖?給鉤體病預(yù)防帶來很大困難。目前普遍使用的鉤體疫苗是根據(jù)當(dāng)?shù)亓餍械臄?shù)種優(yōu)勢(shì)鉤體血清群、型制備的多價(jià)鉤體全菌死疫苗,交叉保護(hù)作用弱且副作用較大。多價(jià)鉤體外膜疫苗是由我國科學(xué)家首先研制成功的組分疫苗,雖顯示免疫效果好和副作用小等優(yōu)點(diǎn),但交叉保護(hù)作用較差的缺陷仍未得到改變。位于鉤體外膜上的外膜蛋白(Outer membrane proteins,OMPs)在決定抗原特異性方面有重要作用,其中一些蛋白為鉤體屬特異性抗原,例如LipL41,它們的編碼基因幾乎保守的存在于所有致病鉤體中,在問號(hào)鉤體基因工程疫苗的研制上有廣泛的前景。 外膜脂蛋白LipL41由355個(gè)氨基酸組成,在鉤體的內(nèi)外兩層膜中都有分布,并暴露于外膜表面。Haake等(2000)克隆出lipL41全長(zhǎng)基因序列,并發(fā)現(xiàn)它只存在于致病鉤體中,而在非致病鉤體中則測(cè)不到。我們以往的研究結(jié)果也證實(shí)我國15群15型問號(hào)鉤體標(biāo)準(zhǔn)參考株均有l(wèi)ipL41基因,但存在兩個(gè)不同的基因型lipL41/1和lipL41/2,其中l(wèi)ipL41/1基因型包含了我國人群中流行最廣的問號(hào)鉤體黃疸出血群、波摩那群、流感傷寒群、秋季群和賽羅群等,而lipL41/2基因型所包含的鉤體血清群在國內(nèi)的發(fā)病率都較低。此外研究還發(fā)現(xiàn)多數(shù)問號(hào)鉤體臨床分離株都含有ipL41基因,并且表達(dá)LipL41蛋白,在多數(shù)鉤體病人的血清中也可測(cè)到相應(yīng)保護(hù)性抗體的存在。 含多個(gè)和多種抗原表位(epitope)的多抗原肽(multiple antigenicpeptide,MAP)是近年發(fā)展起來的一種研制新型基因工程疫苗的先進(jìn)技術(shù)和研究策略,是解決普通鉤體疫苗交叉保護(hù)作用弱的有效途徑,而鑒定有效的抗原表位是研制MAP的第一步。研究抗原表位不僅可深層次的揭示蛋白抗原分子誘導(dǎo)免疫應(yīng)答的分子機(jī)制及特點(diǎn),而且在研制新型分子疫苗方面也有重要應(yīng)用前景。通過噬菌體展示系統(tǒng)對(duì)LipL41/1和LipL41/2的T和B有效聯(lián)合表位進(jìn)行鑒定,可為進(jìn)一步研制鉤體MAP疫苗奠定基礎(chǔ);序列對(duì)比發(fā)現(xiàn)兩個(gè)基因型存在少量氨基酸序列差異,且某些殘基的差異使兩個(gè)基因型的某些預(yù)測(cè)表位產(chǎn)生了較為顯著的差異,對(duì)這些有差異的表位進(jìn)行鑒定有利于深入了解其抗原性和免疫反應(yīng)性特點(diǎn)及個(gè)別氨基酸序列突變對(duì)其表位抗原性的影響,同時(shí)也可為下一步評(píng)估MAP疫苗的保護(hù)范圍提供參考。 實(shí)驗(yàn)方法采用生物信息學(xué)技術(shù)對(duì)LipL41/1和LipL41/2的T細(xì)胞和B細(xì)胞表位進(jìn)行預(yù)測(cè)和分析。選擇主要T細(xì)胞和B細(xì)胞聯(lián)合表位肽并融合表達(dá)于M13KE噬菌體PⅢ蛋白上,PEG/NaCl沉淀法提純重組噬菌體,SDS-PAGE鑒定目的重組PⅢ蛋白(rPⅢ)。分別以自制備rLipL41/1、rLipL41/2、黃疸出血群賴型賴株血清和病人抗血清為一抗,采用Western blot對(duì)上述表位肽進(jìn)行鑒定和篩選。 結(jié)果通過抗原表位預(yù)測(cè),選擇了6個(gè)LipL41/1和2個(gè)LipL41/2聯(lián)合表位。經(jīng)擴(kuò)增獲得了預(yù)期的各抗原表位片段。各目的表位序列均準(zhǔn)確插入噬菌體PⅢ蛋白N端并有效表達(dá)。各抗血清均能識(shí)別上述8個(gè)聯(lián)合表位,但雜交信號(hào)強(qiáng)度存在差異,表現(xiàn)在兩個(gè)方面:一是用同一抗血清為一抗時(shí),不同表位的雜交信號(hào)強(qiáng)度不同,另一方面是同一表位對(duì)不同抗血清的相對(duì)信號(hào)強(qiáng)度也存在差異。表位中或附近的氨基酸序列突變可能會(huì)對(duì)表位的抗原性產(chǎn)生一定的影響,既可以使突變后表位與原抗體的親和力升高也可表現(xiàn)為降低。綜合8個(gè)表位對(duì)不同抗血清的Western Blot結(jié)果及實(shí)際意義,表位LipL41/1-30和LipL41/1-233的信號(hào)強(qiáng)度在兔抗全菌血清和多數(shù)病人血清中的雜交信號(hào)強(qiáng)度均較強(qiáng),其余表位的信號(hào)強(qiáng)度較弱或變異較大。 結(jié)論本研究成功構(gòu)建了T細(xì)胞和B細(xì)胞聯(lián)合表位肽段的噬菌體展示系統(tǒng),所采用的生物信息學(xué)技術(shù)可有效的預(yù)測(cè)抗原表位。實(shí)驗(yàn)鑒定所構(gòu)建的8個(gè)表位均為L(zhǎng)ipL41的有效抗原表位,其中表位LipL41/1-30和LipL41/1-233可應(yīng)用于鉤體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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

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