蚯蚓与日本血吸虫免疫交叉反应分子机制的研究

发布时间：2018-04-20 21:31

本文选题：蚯蚓 + 日本血吸虫　；参考：《华中科技大学》2006年硕士论文

【摘要】： 目的免疫学检测方法的准确性主要取决于诊断抗原的性质,目前用于日本血吸虫病血清免疫学诊断的抗原主要为日本血吸虫(Schistosoma japonicum,Sj)成虫或虫卵的粗抗原,由于抗原组分复杂,与临床常见寄生蠕虫间交叉反应多,严重影响诊断的准确性[1-8]。为了解决这一交叉反应难题,不少学者主要致力于寻找敏感性和特异性兼备的诊断抗原分子,而对交叉反应的机理研究相对甚少。1995年Wisnewski[9]报道:陆生蚯蚓(Lumbricus terrestris,Lt)抗原能与血吸虫病患者血清结合,而不与正常人血清反应;竞争性ELISA抑制水平可达30%以上;用Lt抗原免疫感染血吸虫的小鼠可获得36%减虫率,产生的抗体能与多种血吸虫成虫抗原结合。这一研究结果,为寻找常见寄生蠕虫间交叉反应的同源性抗原分子提供了一条新思路。本课题组多年来在对蚯蚓与血吸虫、肺吸虫、囊虫和旋毛虫等常见寄生蠕虫间交叉反应现象的研究[10-13]中发现:蚯蚓抗原能降低血吸虫病、肺吸虫病、囊虫病、旋毛虫病等蠕虫病患者血清抗体相互之间的交叉反应率;目前还没有某种蠕虫抗原能象蚯蚓抗原一样,同时识别血吸虫病、肺吸虫病、旋毛虫病和囊虫病患者血清,而不与正常人血清发生反应。鉴此,蚯蚓作为一种特殊抗原在血吸虫病诊断研究中有着潜在的应用价值。本实验采用噬菌体展示技术(phage display technique, PDT)筛选获得蚯蚓与日本血吸虫的共同模拟表位;通过饱和硫酸铵溶液梯度盐析法提取蚯蚓可溶性蛋白;利用SDS-PAGE和Western-blot技术,对蚯蚓与日本血吸虫共同模拟表位、蚯蚓的梯度盐析蛋白、蚓激酶(lumbrukinase)和应激蛋白(Stress proteins , SP)等分子成分及交叉反应性进行分析,探讨蚯蚓与日本血吸虫间交叉反应的重要分子基础,为研究和解决临床常见蠕虫病血清免疫学诊断中交叉反应现象提供理论和实验依据。方法 1.蚯蚓与日本血吸虫共同模拟表位的筛选及交叉反应性分析 (1)蚯蚓与日本血吸虫共同模拟表位的筛选依次以日本血吸虫病患者血清(Sj-Ig)、蚯蚓免疫兔血清(Lt-Ig)和Lt-Ig、Sj-Ig作为靶分子,以噬菌体12肽库的第三轮扩增肽库为源肽库,进行2轮吸附-洗脱-扩增的免疫筛选,每轮随机挑取蓝色噬斑各21个,ELISA方法检测其抗原性,并对其反应性较好的阳性克隆进行测序。 (2)共同模拟表位交叉反应性的分析采用SDS-PAGE和Western-blot方法,制备12%分离胶和6%浓缩胶,对经筛选所获的蚯蚓与日本血吸虫共同模拟表位进行SDS-PAGE电泳,分析蛋白组分。电泳分离后转至NC膜,并与日本血吸虫病患者血清反应,分析蛋白组分的交叉反应性。 2.蚯蚓梯度盐析蛋白的制备及交叉反应性分析 (1)蚯蚓梯度盐析蛋白的制备参考饱和硫酸铵溶液盐析提取蚯蚓蚓激酶的方法[14-17],依次用30%、40%、50%、60%、70%饱和硫酸铵溶液梯度盐析,沉淀蚯蚓可溶性蛋白。 (2)盐析蛋白交叉反应性的分析采用SDS-PAGE和Western-blot,制备12%分离胶和6%浓缩胶,对蚯蚓的盐析蛋白进行SDS-PAGE电泳,分析蛋白组分。电泳分离后转至NC膜,并与日本血吸虫病患者血清反应,分析蛋白组分的交叉反应性。 3.蚯蚓蚓激酶交叉反应性的分析采用SDS-PAGE和Western-blot,具体操作同蚯蚓梯度盐析蛋白交叉反应性的分析。 4.蚯蚓应激蛋白的制备及交叉反应性分析 (1)蚯蚓应激蛋白的制备参考加热刺激产生应激蛋白的相关文献[18-21],取蚯蚓10条,40℃加热30 min,收集蚯蚓分泌物,即蚯蚓应激蛋白。 (2)应激蛋白交叉反应性的分析采用SDS-PAGE和Western-blot,具体操作同蚯蚓梯度盐析蛋白交叉反应性的分析。结果 1.蚯蚓与日本血吸虫共同模拟表位的筛选及交叉反应性分析 (1)共同模拟表位的筛选经过2轮的吸附-洗脱-扩增,获得了6个A491nm值较高的克隆(Lt9、Lt14、Sj17、Sj18、SjLt6、SjLt11),通过DNA测序,经BLAST软件分析,发现:Lt9与血吸虫的抱雌沟蛋白和蚯蚓的NADH脱氢酶亚单位1有4个连续的氨基酸(LAET)一致;Sj17与血吸虫的1-α延长因子和蚯蚓的1-α延长因子有4个不连续氨基酸(HT-HI)一致;SjLt11与血吸虫的乳酸脱氢酶样蛋白和蚯蚓的NADH脱氢酶亚单位6有4个连续的氨基酸(NSIL)一致。 (2) 3个共同模拟表位交叉反应性的分析将Lt9、Sj17、SjLt11这3个共同模拟表位同日本血吸虫病患者血清反应,得到分子量为64.9 kDa的交叉反应带。 2.蚯蚓梯度盐析蛋白的SDS-PAGE和Western-blot结果30%～70%饱和(NH4)2SO4溶液能沉淀较多的蚯蚓可溶性蛋白,对应的分子量在25.3～112.7kDa和18.3kDa范围内,其中主带分子量在25.3～54.7kDa和18.3kDa;与日本血吸虫病患者血清反应,对应显色带的分子量在20.4～110.2kDa,其中主带分子量为34.7、37.1～45.2、59.2、76.7～81.9、88.4～96.6 kDa。 3.蚯蚓蚓激酶的SDS-PAGE和Western-blot结果在SDS-PAGE胶上存在分子量为26.7～97.8 kDa的蛋白带,与日本血吸虫病患者血清反应,对应显色带的分子量在39.8～95.1 kDa。 4.蚯蚓应激蛋白的SDS-PAGE和Western-blot结果在SDS-PAGE胶上存在分子量为28.3～97.8kDa和19.6 kDa的蛋白带,与日本血吸虫病患者血清反应,对应显色带的分子量在61.2～95.1 kDa。结论 1.用日本血吸虫病患者血清和蚯蚓免疫兔血清筛选噬菌体12肽库可获得3个蚯蚓与日本血吸虫共同模拟表位。 2.饱和硫酸铵溶液梯度盐析法能有效地从蚯蚓可溶性物质中提取到与日本血吸虫间交叉反应的蛋白抗原组分,对应的分子量在20.4～110.2kDa。 3.蚓激酶与日本血吸虫间存在交叉反应抗原,对应的分子量在39.8～95.1 kDa。 4.蚯蚓应激蛋白与日本血吸虫间也存在交叉反应抗原,对应的分子量在61.2～95.1 kDa。综上所述,蚯蚓与日本血吸虫共同模拟表位、蚯蚓的盐析蛋白、蚓激酶和应激蛋白与日本血吸虫病患者血清间均存在分子量为61.2～95.1 kDa的免疫交叉反应带,表明:蚯蚓与日本血吸虫间存在分子量为61.2～95.1 kDa的共同蛋白组分,该组分中可能部分属于酶类,氨基酸序列可能包含NSIL、LAET和HT-HI片段,该类物质是蚯蚓与日本血吸虫免疫交叉反应的重要分子基础。由此可见,蚯蚓与日本血吸虫间免疫交叉反应的分子基础可能部分属于酶类,蚯蚓可溶性蛋白作为一种特殊抗原在寄生虫病诊断研究中有着广阔的应用前景。
[Abstract]:objective
The accuracy of immunological detection methods mainly depends on the nature of the diagnostic antigen. The antigen used to diagnose the serum immunology of schistosomiasis in Japan is mainly the Schistosoma japonicum (Sj) adult or the crude antigen of the eggs. Because the antigen composition is complex, the cross reaction between the common parasitic worms and the common parasitic worms is more serious, which seriously affects the diagnosis. Accuracy [1-8].
In order to solve this cross reaction problem, many scholars have focused on finding sensitive and specific diagnostic antigen molecules, and the mechanism of cross reaction is relatively rare in.1995 year Wisnewski[9] report: Lumbricus terrestris (Lt) antigen can be combined with serum of patients with blood fluke disease, but not with normal human serum. The inhibitory level of competitive ELISA can reach more than 30%; the mice infected with Schistosoma with Lt antigen can obtain the rate of 36% worm reduction, and the antibody produced can be combined with the antigen of a variety of Schistosoma japonicum. This study provides a new idea for finding homologous antigens of the common parasitic intersecting cross reactions.
The study of the cross reaction between earthworms and Schistosoma, paragonimiasis, cysticercosis and Trichinella in [10-13] has been found in the research group for many years. The earthworm antigen can reduce the cross reaction rate of sera antibodies against schistosomiasis, paragonimiasis, cysticercosis, trichinosis and other worms. There is no worm at present. Antigens, like earthworm antigens, recognize both schistosomiasis, paragonimiasis, trichinosis, and cysticercosis, but do not react with normal human serum.
Therefore, earthworm as a special antigen has potential application in the diagnosis of schistosomiasis.
In this experiment, the common simulated epitopes of earthworms and Schistosoma japonicum were obtained by phage display technique (PDT), and the soluble protein of earthworms was extracted by the gradient salting out method of saturated ammonium sulfate solution. The common simulated epitopes of earthworms and Schistosoma japonicum and the gradient salting of earthworms by SDS-PAGE and Western-blot technology were used. The molecular components and cross reactivity of lumbrukinase and Stress proteins (SP) were analyzed to explore the important molecular basis of cross reaction between earthworms and Schistosoma japonicum, and to provide theoretical and experimental basis for the study and solution of cross reaction in serum immunological diagnosis of common vermicrasis.
Method
Screening and cross reactivity analysis of 1. mimic epitopes of Schistosoma japonicum and Schistosoma japonicum
(1) screening of mimic epitopes of Schistosoma japonicum and earthworms
The sera (Sj-Ig), earthworm immune rabbit serum (Lt-Ig) and Lt-Ig, Sj-Ig were used as the target molecules in turn, and the third round amplification peptide library of phage 12 peptide library was used as the source peptide library, and the 2 round of adsorption elution amplification immunization was carried out, and 21 blue plaques were selected randomly each round. The ELISA method was used to detect its antigenicity and its reactivity was compared. Good positive clones are sequenced.
(2) analysis of the cross reactivity of the common simulated epitopes
The SDS-PAGE and Western-blot methods were used to prepare 12% separation gel and 6% concentrated glue. SDS-PAGE electrophoresis was carried out for the common simulated epitopes of earthworms and Schistosoma japonicum. The protein components were analyzed and then transferred to the NC membrane by electrophoresis and the cross reactivity of the protein components in the sera of schistosomiasis japonica.
Preparation and cross reactivity analysis of 2. earthworm gradient salting out protein
(1) preparation of earthworm gradient salting out protein in the preparation of the method [14-17] for extracting earthworm kinase from saturated ammonium sulfate solution by reference saturated ammonium sulfate solution. The solution of earthworm soluble protein was precipitated by gradient salting out in 30%, 40%, 50%, 60%, 70% saturated ammonium sulfate solution in turn.
(2) analysis of the cross reactivity of salting out protein
SDS-PAGE and Western-blot were used to prepare 12% separation glue and 6% concentrated glue. The salted out protein of earthworm was electrophoretic and the protein component was analyzed. After electrophoresis, it was transferred to NC membrane and reacted with the sera of schistosomiasis japonica and analyzed the cross reactivity of the protein components.
Analysis of cross reactivity of earthworm kinase 3.
SDS-PAGE and Western-blot were used to analyze the cross reactivity of gradient salting out protein with earthworm.
Preparation and cross reactivity analysis of 4. earthworm stress proteins
(1) the related literature of stress protein produced by earthworm stress protein was prepared by reference heating to produce stress protein, including 10 worms and 30 min heating at 40 C to collect earthworm secretions, that is, earthworm stress protein ([18-21]).
(2) analysis of the cross reactivity of stress protein
SDS-PAGE and Western-blot were used to analyze the cross reactivity of gradient salting out protein with earthworm.
Result
Screening and cross reactivity analysis of 1. mimic epitopes of Schistosoma japonicum and Schistosoma japonicum
(1) screening of common simulated epitopes
After 2 rounds of adsorption - elution - amplification, 6 high A491nm clones (Lt9, Lt14, Sj17, Sj18, SjLt6, SjLt11) were obtained. Through DNA sequencing, the BLAST software analysis showed that Lt9 and Schistosoma of Schistosoma and the NADH dehydrogenase subunit 1 of the worms were consistent with 4 consecutive amino acids (LAET), and the alpha prolongation factor and earthworm of Schistosoma japonicum. The 1- alpha lengthening factor is consistent with 4 discontinuous amino acids (HT-HI); SjLt11 and the lactate dehydrogenase like protein of Schistosoma and the NADH dehydrogenase subunit 6 of the earthworm have 4 consecutive amino acids (NSIL).
(2) analysis of the cross reactivity of 3 common simulated epitopes
The 3 common mimic epitopes of Lt9, Sj17 and SjLt11 were reacted with sera from schistosomiasis japonica patients, and the cross reaction band with molecular weight of 64.9 kDa was obtained.
2. the SDS-PAGE and Western-blot of the earthworm gradient salting out protein results in the 30% ~ 70% saturated (NH4) 2SO4 solution that can precipitate more earthworm soluble proteins. The corresponding molecular weight is within the range of 25.3 to 112.7kDa and 18.3kDa, and the molecular weight of the main band is 25.3 to 54.7kDa and 18.3kDa, and the reaction with the sera of the patients with Japanese schistosomiasis corresponds to the molecules of the color zone. The quantity is 20.4 ~ 110.2kDa, and the molecular weight of the main band is 34.7,37.1 ~ 45.2,59.2,76.7 ~ 81.9,88.4 ~ 96.6 kDa..
3. SDS-PAGE and Western-blot results of earthworm kinase
There was a protein band of 26.7 to 97.8 kDa with a molecular weight of SDS-PAGE, which corresponded to the serum of schistosomiasis japonica patients, corresponding to the molecular weight of the color band from 39.8 to 95.1 kDa..
4. the SDS-PAGE and Western-blot results of earthworm stress protein were found on SDS-PAGE gum with a molecular weight of 28.3 to 97.8kDa and 19.6 kDa, and the response to the sera of patients with schistosomiasis, corresponding to the molecular weight of the color bands of 61.2 to 95.1 kDa..
conclusion
1. screening of phage 12 peptide library with Schistosoma japonicum serum and earthworm immune serum from rabbits can obtain 3 mimic epitopes of Schistosoma japonicum and Schistosoma japonicum.
The gradient salting out method of 2. saturated ammonium sulfate solution can be effectively extracted from the soluble substance of earthworm to the protein antigen component of the cross reaction with Schistosoma japonicum. The corresponding molecular weight is from 20.4 to 110.2kDa..
3. there is cross reaction antigen between earthworm kinase and Schistosoma japonicum, and the corresponding molecular weight is between 39.8 and 95.1 kDa..
4. there was cross reaction antigen between the earthworm stress protein and Schistosoma japonicum, and the corresponding molecular weight was between 61.2 and 95.1 kDa..
To sum up, earthworms and Schistosoma japonicum co mimic the epitopes, earthworms' salting proteins, vermis and stress proteins and the sera of patients with schistosomiasis have an immune cross reaction zone with a molecular weight of 61.2 to 95.1 kDa, indicating that there is a common protein component with a molecular weight of 61.2 to 95.1 kDa between earthworms and Schistosoma japonicum, which is a component of this component. Maybe part of the enzyme belongs to the enzyme, and the amino acid sequence may contain NSIL, LAET and HT-HI fragments. This kind of substance is an important molecular basis for the immune cross reaction between earthworms and Schistosoma japonicum.
It can be seen that the molecular basis of the cross reaction between earthworms and Schistosoma japonicum may be part of the enzymes. As a special antigen, the earthworm soluble protein has a broad application prospect in the diagnosis and study of parasitic diseases.

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R383;R392

【参考文献】