本文關(guān)鍵詞：日本血吸蟲29KD膜外蛋白抗體檢測的價值及其單克隆抗體的制備與鑒定　出處：《安徽醫(yī)科大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 日本血吸蟲 膜蛋白 免疫診斷 單克隆抗體

【摘要】： 目的為尋找新的血吸蟲病免疫診斷候選抗原分子,對日本血吸蟲29KD表膜蛋白(Sj29)的膜外區(qū)進行體外重組表達、純化及免疫定位,通過初步的實驗室檢測驗證日本血吸蟲重組表膜蛋白rSj29用于日本血吸蟲病抗體檢測的特異性、敏感性及療效考核所具有的實用價值。同時用純化的重組蛋白免疫小鼠,制備及鑒定單克隆抗體,初步建立抗體夾心ELISA法,擬進一步探討該單克隆抗體用于血吸蟲病人血清循環(huán)抗原檢測的應(yīng)用價值。方法以日本血吸蟲童蟲cDNA文庫為模板,擴增目的基因,構(gòu)建Sj29的膜外區(qū)的原核表達系統(tǒng),在大腸桿菌中獲得了融合表達,用親和層析法制備純化的rSj29膜外蛋白;用急性、慢性和晚期日本血吸蟲患者血清和正常人血清進行Western blotting鑒定。用血吸蟲成蟲冰凍切片以抗rSj29蛋白小鼠免疫血清作為一抗行間接免疫熒光實驗觀察Sj29在日本血吸蟲成蟲的免疫定位。在確定重組蛋白抗原包被濃度、血清稀釋度以及酶標(biāo)抗體工作濃度的基礎(chǔ)上,用rSj29-ELISA和成蟲粗抗原(AWA)-ELISA兩種方法平行檢測35例急性、38例慢性和27例晚期日本血吸蟲病人血清、27例華支睪吸蟲感染者、29例鉤蟲感染者和30例正常對照者血清中抗體,比較不同方法之間的敏感性,特異性以及交叉反應(yīng)性。另外,還用rSj29蛋白免疫小鼠6周后,將其脾細胞與SP2/0骨髓瘤細胞進行雜交融合,HAT篩選出雜交瘤細胞,經(jīng)亞克隆及擴大培養(yǎng),建立能穩(wěn)定分泌抗日本血吸蟲膜蛋白Sj29單克隆抗體的雜交瘤細胞株。用間接ELISA法檢測雜交瘤上清和小鼠腹水效價。采用Sigma抗體亞型檢測試劑盒測定單克隆抗體的Ig類別和亞型。Western-Blotting實驗鑒定其免疫學(xué)特性。用血吸蟲成蟲冰凍切片以抗rSj29蛋白單克隆抗體的小鼠腹水作為一抗行間接免疫熒光實驗觀察2株單克隆抗體在日本血吸蟲成蟲的識別位點。用棋盤滴定法同時選擇包被用抗rSj29兔多克隆抗體、抗rSj29小鼠單克隆抗體以及HRP-山羊抗小鼠抗體的工作濃度。結(jié)果成功克隆并用原核系統(tǒng)表達rSj29蛋白,純化的rSj29膜外蛋白用Western blotting證實能夠被急性、慢性和晚期日本血吸蟲感染患者血清識別而與正常人血清無反應(yīng)。免疫熒光定位結(jié)果證實了生物信息學(xué)的推測——Sj29蛋白是日本血吸蟲成蟲表膜蛋白,該蛋白在日本血吸蟲成蟲的體被表達非常豐富。rSj29-ELISA和AWA-ELISA兩種方法平行檢測急、慢性和晚期血吸蟲病患者,華支睪吸蟲患者,腸道線蟲患者以及正常人血清抗體,結(jié)果為: rSj29-ELISA法檢測急性、慢性和晚期日本血吸蟲病患者血清抗體的敏感性分別為91.4%、89.5%和96.3%,與AWA-ELISA(91.4%、89.5%和88.9%)相比,兩者敏感性統(tǒng)計學(xué)分析上無顯著性差異;檢測鉤蟲、華支睪吸蟲感染者血清抗體的結(jié)果顯示rSj29-ELISA(17.2%、8.1%)與AWA-ELISA(27.6%、18.5%)相比前者的交叉反應(yīng)率絕對值較后者低,但統(tǒng)計學(xué)上無顯著性差異,需加大樣本量進一步確認;檢測日本血吸蟲感染治療半年后患者血清抗體的陰轉(zhuǎn)率兩者相比有統(tǒng)計學(xué)顯著性意義,前者(55.9%)的陰轉(zhuǎn)率明顯比后者(8.9%)的高,說明rSj29-ELISA可考慮作為療效考核的重要參考指標(biāo)之一。與此同時獲得兩株特異性分泌抗Sj29單克隆抗體的雜交瘤細胞株,經(jīng)過體外多次傳代培養(yǎng)后,仍能分泌高效價的抗體,細胞上清效價穩(wěn)定在1:800~1:3200之間。用BALB/c小鼠制備單抗腹水,ELISA法測效價在1.024×105以上。兩株雜交瘤細胞的抗體亞型均為IgG1。Western-Blotting證實兩株細胞均為分泌抗Sj29單克隆抗體的雜交瘤細胞。免疫熒光實驗結(jié)果顯示兩株雜交瘤細胞抗體的識別位點在日本血吸蟲成蟲的體被表膜上。初步建立了雙抗體夾心ELISA用于血吸蟲病人血清循環(huán)抗原檢測的實驗方法。結(jié)論本研究獲得了預(yù)期的rSj29膜外區(qū)融合蛋白,證實了該重組蛋白具有較好的免疫反應(yīng)性和免疫原性,間接免疫熒光結(jié)果顯示Sj29是日本血吸蟲成蟲表膜蛋白。以rSj29膜外區(qū)融合蛋白包被建立了rSj29-ELISA方法用于血吸蟲病人血清循環(huán)抗體的檢測,獲得較好的敏感性和特異性,尤其在療效考核方面比AWA-ELISA更具優(yōu)越性。該重組抗原制備簡便,成本低廉,制備周期短,方法易于標(biāo)準化,更適合商品化生產(chǎn)和流行區(qū)血吸蟲病的血清學(xué)輔助診斷。同時獲得了2株特異性分泌抗Sj29單克隆抗體的細胞株,且細胞上清和腹水中單克隆抗體的效價均較高。兩株單抗亞型均為IgG1。Western-Blotting結(jié)果證實了該2株單克隆抗體的免疫學(xué)特性,免疫熒光實驗結(jié)果證實兩株雜交瘤細胞抗體的識別位點位于日本血吸蟲成蟲的體被表膜上。初步建立了雙抗體夾心ELISA方法用于檢測日本血吸蟲病人血清中循環(huán)抗原。
[Abstract]:Objective to find a new candidate antigen for immunodiagnosis of schistosomiasis. Schistosoma japonicum 29KD membrane protein (Sj29) of the extracellular domain of recombinant expression, purification and immunological localization, through preliminary laboratory testing verification of Schistosoma japonicum recombinant membrane protein rSj29 for the detection of Schistosoma japonicum antibody specificity, sensitivity and efficacy evaluation of practical value. Meanwhile, the purified recombinant protein was used to immunize mice, preparation and identification of monoclonal antibodies. The sandwich ELISA method was initially established to further explore the application value of the monoclonal antibody for detection of circulating antigen in serum of patients with schistosomiasis. Methods Schistosoma japonicum cDNA library as template, PCR, prokaryotic expression system to construct the Sj29 extracellular domain in Escherichia coli, obtained fusion protein by affinity chromatography preparation of purified rSj29 proteins were identified by blotting Western; acute, chronic and advanced Japanese blood sucking insects and serum normal human serum. The frozen sections of Schistosoma japonicum adults were used to detect the immunization location of Sj29 in adult worms of Japanese Schistosoma japonicum using anti rSj29 protein immunized serum as an indirect immunofluorescence assay. In determining the recombinant protein antigen concentration, serum dilution and the enzyme labeled antibody concentration on the basis of rSj29-ELISA and adult worm antigen (AWA) detected 35 cases of acute and chronic 38 cases and 27 cases of advanced schistosomiasis patients serum, 27 cases of infection, 29 cases of Clonorchis sinensis infected with hookworm and 30 normal controls in the serum antibody -ELISA two methods, the sensitivity comparison between different methods, specificity and cross reactivity. In addition, the mice were immunized with rSj29 protein for 6 weeks, and then their spleen cells were fused with SP2/0 myeloma cells. HAT hybridoma cells were screened out, and hybridoma cell lines secreting monoclonal antibodies against Schistosoma japonicum membrane protein Sj29 were established by subcloning and expansion culture. Indirect ELISA was used to detect the titer of hybridoma supernatant and mouse ascites. The Sigma antibody subtype detection kit was used to determine the Ig category and subtype of the monoclonal antibody. The immunological characteristics were identified by Western-Blotting experiment. We used frozen section of Schistosoma japonicum adult worms and anti ascites of anti rSj29 protein monoclonal antibody as an indirect immunofluorescence assay to observe the identification sites of 2 monoclonal antibodies in adult worms of Schistosoma japonicum. The working concentration of anti rSj29 rabbit polyclonal antibody, anti rSj29 mouse monoclonal antibody and HRP- Goat anti mouse antibody was selected by chessboard titration. Results rSj29 protein was successfully cloned and expressed in prokaryotic system. The purified rSj29 outer membrane protein was identified by Western blotting, and it could be identified by serum from acute, chronic and advanced schistosomiasis patients, but not from normal human serum. Immunofluorescence localization results confirm the bioinformatics speculation that Sj29 protein is the membrane protein of Schistosoma japonicum adult, which is abundant in the body of Schistosoma japonicum adult. RSj29-ELISA and AWA-ELISA two parallel detection method of acute and chronic and advanced schistosomiasis, clonorchiasis patients, intestinal nematode patients and normal human serum antibody. The results are as follows: the sensitivity of rSj29-ELISA detection of acute, chronic and advanced schistosomiasis patients serum antibody were 91.4%, 89.5% and 96.3%, and (91.4%, 89.5% and AWA-ELISA 88.9%), there was no significant difference between the sensitivity of statistical analysis on the detection results of serum antibody; hookworm, Clonorchis sinensis infection shows that rSj29-ELISA (17.2%, 8.1%) and AWA-ELISA (27.6%, 18.5%) compared with the cross reaction rate of the former absolute value is smaller than the latter, but there are no statistically significant differences, the need to increase the sample the amount of further confirmation; detection of Schistosoma japonicum infection compared to serum antibody in patients with negative rate of two half a year after treatment was statistically significant significance, the former (55.9%). The rate of negative conversion was significantly higher than that of the latter (8.9%), indicating that rSj29-ELISA could be considered as one of the important reference indexes for the assessment of curative effect. At the same time, two hybridoma cell lines secreting anti Sj29 monoclonal antibodies were obtained. After repeated passages in vitro, they could still secrete highly effective antibodies, and the titer of cell supernatants was stable between 1:800~1:3200. The McAb ascites were prepared by BALB/c mice, and the titer of ELISA was over 1.024 * 105. The antibody subtypes of the two hybridoma cells were all IgG1. Western-Blotting confirmed that all of the two cells were hybridoma cells that secreted anti Sj29 monoclonal antibodies. The results of immunofluorescence test showed that the identification site of two hybridoma cell antibodies was on the surface of the body of the adult Schistosoma japonicum. An experimental method for the detection of circulating antigen in sera of Schistosoma japonicum by double antibody sandwich ELISA was preliminarily established. Conclusion the expected rSj29 outer membrane fusion protein has been obtained. It is confirmed that the recombinant protein has good immunogenicity and immunogenicity. Indirect immunofluorescence results indicate that Sj29 is the membrane protein of Schistosoma japonicum adult. A rSj29-ELISA method was developed for the detection of circulating antibodies in sera of schistosomiasis patients by the inclusion of rSj29 outer membrane fusion protein. It has good sensitivity and specificity, especially in the aspect of efficacy evaluation, which is more advantageous than AWA-ELISA. The recombinant antigen is easy to prepare, inexpensive, short in preparation cycle, and easy to standardize. It is more suitable for commercial production and serological diagnosis of schistosomiasis in epidemic area. At the same time, 2 cell lines that specifically secreted anti Sj29 monoclonal antibodies were obtained, and the potency of monoclonal antibodies in cell supernatant and ascites was higher. The subtypes of two mAb were all IgG1. Western-Blotting results confirmed the immunological characteristics of the 2 monoclonal antibodies. Immunofluorescence results confirmed that the identification sites of the two hybridoma antibodies were located on the body surface membrane of adult Schistosoma japonicum. A double antibody sandwich ELISA method was initially established
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392

【引證文獻】

相關(guān)碩士學(xué)位論文 前1條

1 王萍;日本血吸蟲重組膜外蛋白rSj29免疫診斷的初步應(yīng)用及代謝抗原單克隆抗體的制備[D];安徽醫(yī)科大學(xué);2010年

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本文編號：1343380