本文選題：蚯蚓 + 日本血吸蟲　； 參考：《華中科技大學》2006年碩士論文

【摘要】： 目的 免疫學檢測方法的準確性主要取決于診斷抗原的性質(zhì),目前用于日本血吸蟲病血清免疫學診斷的抗原主要為日本血吸蟲(Schistosoma japonicum,Sj)成蟲或蟲卵的粗抗原,由于抗原組分復雜,與臨床常見寄生蠕蟲間交叉反應多,嚴重影響診斷的準確性[1-8]。 為了解決這一交叉反應難題,不少學者主要致力于尋找敏感性和特異性兼?zhèn)涞脑\斷抗原分子,而對交叉反應的機理研究相對甚少。1995年Wisnewski[9]報道:陸生蚯蚓(Lumbricus terrestris,Lt)抗原能與血吸蟲病患者血清結(jié)合,而不與正常人血清反應;競爭性ELISA抑制水平可達30%以上;用Lt抗原免疫感染血吸蟲的小鼠可獲得36%減蟲率,產(chǎn)生的抗體能與多種血吸蟲成蟲抗原結(jié)合。這一研究結(jié)果,為尋找常見寄生蠕蟲間交叉反應的同源性抗原分子提供了一條新思路。 本課題組多年來在對蚯蚓與血吸蟲、肺吸蟲、囊蟲和旋毛蟲等常見寄生蠕蟲間交叉反應現(xiàn)象的研究[10-13]中發(fā)現(xiàn):蚯蚓抗原能降低血吸蟲病、肺吸蟲病、囊蟲病、旋毛蟲病等蠕蟲病患者血清抗體相互之間的交叉反應率;目前還沒有某種蠕蟲抗原能象蚯蚓抗原一樣,同時識別血吸蟲病、肺吸蟲病、旋毛蟲病和囊蟲病患者血清,而不與正常人血清發(fā)生反應。 鑒此,蚯蚓作為一種特殊抗原在血吸蟲病診斷研究中有著潛在的應用價值。 本實驗采用噬菌體展示技術(shù)(phage display technique, PDT)篩選獲得蚯蚓與日本血吸蟲的共同模擬表位;通過飽和硫酸銨溶液梯度鹽析法提取蚯蚓可溶性蛋白;利用SDS-PAGE和Western-blot技術(shù),對蚯蚓與日本血吸蟲共同模擬表位、蚯蚓的梯度鹽析蛋白、蚓激酶(lumbrukinase)和應激蛋白(Stress proteins , SP)等分子成分及交叉反應性進行分析,探討蚯蚓與日本血吸蟲間交叉反應的重要分子基礎(chǔ),為研究和解決臨床常見蠕蟲病血清免疫學診斷中交叉反應現(xiàn)象提供理論和實驗依據(jù)。 方法 1.蚯蚓與日本血吸蟲共同模擬表位的篩選及交叉反應性分析 (1)蚯蚓與日本血吸蟲共同模擬表位的篩選 依次以日本血吸蟲病患者血清(Sj-Ig)、蚯蚓免疫兔血清(Lt-Ig)和Lt-Ig、Sj-Ig作為靶分子,以噬菌體12肽庫的第三輪擴增肽庫為源肽庫,進行2輪吸附-洗脫-擴增的免疫篩選,每輪隨機挑取藍色噬斑各21個,ELISA方法檢測其抗原性,并對其反應性較好的陽性克隆進行測序。 (2)共同模擬表位交叉反應性的分析 采用SDS-PAGE和Western-blot方法,制備12%分離膠和6%濃縮膠,對經(jīng)篩選所獲的蚯蚓與日本血吸蟲共同模擬表位進行SDS-PAGE電泳,分析蛋白組分。電泳分離后轉(zhuǎn)至NC膜,并與日本血吸蟲病患者血清反應,分析蛋白組分的交叉反應性。 2.蚯蚓梯度鹽析蛋白的制備及交叉反應性分析 (1)蚯蚓梯度鹽析蛋白的制備參考飽和硫酸銨溶液鹽析提取蚯蚓蚓激酶的方法[14-17],依次用30%、40%、50%、60%、70%飽和硫酸銨溶液梯度鹽析,沉淀蚯蚓可溶性蛋白。 (2)鹽析蛋白交叉反應性的分析 采用SDS-PAGE和Western-blot,制備12%分離膠和6%濃縮膠,對蚯蚓的鹽析蛋白進行SDS-PAGE電泳,分析蛋白組分。電泳分離后轉(zhuǎn)至NC膜,并與日本血吸蟲病患者血清反應,分析蛋白組分的交叉反應性。 3.蚯蚓蚓激酶交叉反應性的分析 采用SDS-PAGE和Western-blot,具體操作同蚯蚓梯度鹽析蛋白交叉反應性的分析。 4.蚯蚓應激蛋白的制備及交叉反應性分析 (1)蚯蚓應激蛋白的制備參考加熱刺激產(chǎn)生應激蛋白的相關(guān)文獻[18-21],取蚯蚓10條,40℃加熱30 min,收集蚯蚓分泌物,即蚯蚓應激蛋白。 (2)應激蛋白交叉反應性的分析 采用SDS-PAGE和Western-blot,具體操作同蚯蚓梯度鹽析蛋白交叉反應性的分析。 結(jié)果 1.蚯蚓與日本血吸蟲共同模擬表位的篩選及交叉反應性分析 (1)共同模擬表位的篩選 經(jīng)過2輪的吸附-洗脫-擴增,獲得了6個A491nm值較高的克隆(Lt9、Lt14、Sj17、Sj18、SjLt6、SjLt11),通過DNA測序,經(jīng)BLAST軟件分析,發(fā)現(xiàn):Lt9與血吸蟲的抱雌溝蛋白和蚯蚓的NADH脫氫酶亞單位1有4個連續(xù)的氨基酸(LAET)一致;Sj17與血吸蟲的1-α延長因子和蚯蚓的1-α延長因子有4個不連續(xù)氨基酸(HT-HI)一致;SjLt11與血吸蟲的乳酸脫氫酶樣蛋白和蚯蚓的NADH脫氫酶亞單位6有4個連續(xù)的氨基酸(NSIL)一致。 (2) 3個共同模擬表位交叉反應性的分析 將Lt9、Sj17、SjLt11這3個共同模擬表位同日本血吸蟲病患者血清反應,得到分子量為64.9 kDa的交叉反應帶。 2.蚯蚓梯度鹽析蛋白的SDS-PAGE和Western-blot結(jié)果30%～70%飽和(NH4)2SO4溶液能沉淀較多的蚯蚓可溶性蛋白,對應的分子量在25.3～112.7kDa和18.3kDa范圍內(nèi),其中主帶分子量在25.3～54.7kDa和18.3kDa;與日本血吸蟲病患者血清反應,對應顯色帶的分子量在20.4～110.2kDa,其中主帶分子量為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結(jié)果 在SDS-PAGE膠上存在分子量為26.7～97.8 kDa的蛋白帶,與日本血吸蟲病患者血清反應,對應顯色帶的分子量在39.8～95.1 kDa。 4.蚯蚓應激蛋白的SDS-PAGE和Western-blot結(jié)果在SDS-PAGE膠上存在分子量為28.3～97.8kDa和19.6 kDa的蛋白帶,與日本血吸蟲病患者血清反應,對應顯色帶的分子量在61.2～95.1 kDa。 結(jié)論 1.用日本血吸蟲病患者血清和蚯蚓免疫兔血清篩選噬菌體12肽庫可獲得3個蚯蚓與日本血吸蟲共同模擬表位。 2.飽和硫酸銨溶液梯度鹽析法能有效地從蚯蚓可溶性物質(zhì)中提取到與日本血吸蟲間交叉反應的蛋白抗原組分,對應的分子量在20.4～110.2kDa。 3.蚓激酶與日本血吸蟲間存在交叉反應抗原,對應的分子量在39.8～95.1 kDa。 4.蚯蚓應激蛋白與日本血吸蟲間也存在交叉反應抗原,對應的分子量在61.2～95.1 kDa。 綜上所述,蚯蚓與日本血吸蟲共同模擬表位、蚯蚓的鹽析蛋白、蚓激酶和應激蛋白與日本血吸蟲病患者血清間均存在分子量為61.2～95.1 kDa的免疫交叉反應帶,表明:蚯蚓與日本血吸蟲間存在分子量為61.2～95.1 kDa的共同蛋白組分,該組分中可能部分屬于酶類,氨基酸序列可能包含NSIL、LAET和HT-HI片段,該類物質(zhì)是蚯蚓與日本血吸蟲免疫交叉反應的重要分子基礎(chǔ)。 由此可見,蚯蚓與日本血吸蟲間免疫交叉反應的分子基礎(chǔ)可能部分屬于酶類,蚯蚓可溶性蛋白作為一種特殊抗原在寄生蟲病診斷研究中有著廣闊的應用前景。
[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.

【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R383;R392

【參考文獻】

相關(guān)期刊論文 前10條

1 陳文強,鄧百萬;氨基酸的置換與生物大分子進化的保守性的評析[J];氨基酸和生物資源;2001年04期

2 柳建發(fā);目前人體寄生蟲病的現(xiàn)狀和研究進展[J];地方病通報;1999年04期

3 胡宗海;低氧與應激蛋白研究進展[J];第三軍醫(yī)大學學報;2003年18期

4 黃京飛,Ｔｏｍ Ｌ．Ｂｌｕｎｄｅlｌ;蛋白質(zhì)序列和結(jié)構(gòu)的保守性與其功能的關(guān)系[J];動物學研究;1999年01期

5 彭先鳳,陶建寧,彭余田,鮑錦庫;赤子愛勝蚓五種纖溶酶組分的分離純化及對纖維蛋白原酶解的初步研究[J];華西藥學雜志;1999年01期

6 郭俊生,趙法,王楓,陳洪章;熱暴露對果蠅壽命及熱應激蛋白(Hsp70)含量的影響[J];解放軍預防醫(yī)學雜志;1998年04期

7 李菁華,趙偉;蚓激酶研究與應用進展[J];錦州醫(yī)學院學報;2004年05期

8 周曉紅,陳曉光,戴琳,胡旭初,李華,劉國章;日本血吸蟲可溶性蟲卵抗原38kDa分子Dipstick快速診斷模式的初步建立[J];熱帶醫(yī)學雜志;2001年01期

9 婁文嫻,吳琛耘,張恩英,薛純良;單克隆抗體夾心ELISA定量檢測日本血吸蟲循環(huán)抗原[J];上海第二醫(yī)科大學學報;1999年03期

10 何執(zhí)中,郭懷芳,唐寧,方成;蚯蚓纖溶酶的分離純化及抗栓活性研究[J];中國生化藥物雜志;2001年06期

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