日本血吸虫29KD膜外蛋白抗体检测的价值及其单克隆抗体的制备与鉴定

发布时间：2017-12-27 22:10

本文关键词：日本血吸虫29KD膜外蛋白抗体检测的价值及其单克隆抗体的制备与鉴定　出处：《安徽医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的为寻找新的血吸虫病免疫诊断候选抗原分子,对日本血吸虫29KD表膜蛋白(Sj29)的膜外区进行体外重组表达、纯化及免疫定位,通过初步的实验室检测验证日本血吸虫重组表膜蛋白rSj29用于日本血吸虫病抗体检测的特异性、敏感性及疗效考核所具有的实用价值。同时用纯化的重组蛋白免疫小鼠,制备及鉴定单克隆抗体,初步建立抗体夹心ELISA法,拟进一步探讨该单克隆抗体用于血吸虫病人血清循环抗原检测的应用价值。方法以日本血吸虫童虫cDNA文库为模板,扩增目的基因,构建Sj29的膜外区的原核表达系统,在大肠杆菌中获得了融合表达,用亲和层析法制备纯化的rSj29膜外蛋白;用急性、慢性和晚期日本血吸虫患者血清和正常人血清进行Western blotting鉴定。用血吸虫成虫冰冻切片以抗rSj29蛋白小鼠免疫血清作为一抗行间接免疫荧光实验观察Sj29在日本血吸虫成虫的免疫定位。在确定重组蛋白抗原包被浓度、血清稀释度以及酶标抗体工作浓度的基础上,用rSj29-ELISA和成虫粗抗原(AWA)-ELISA两种方法平行检测35例急性、38例慢性和27例晚期日本血吸虫病人血清、27例华支睾吸虫感染者、29例钩虫感染者和30例正常对照者血清中抗体,比较不同方法之间的敏感性,特异性以及交叉反应性。另外,还用rSj29蛋白免疫小鼠6周后,将其脾细胞与SP2/0骨髓瘤细胞进行杂交融合,HAT筛选出杂交瘤细胞,经亚克隆及扩大培养,建立能稳定分泌抗日本血吸虫膜蛋白Sj29单克隆抗体的杂交瘤细胞株。用间接ELISA法检测杂交瘤上清和小鼠腹水效价。采用Sigma抗体亚型检测试剂盒测定单克隆抗体的Ig类别和亚型。Western-Blotting实验鉴定其免疫学特性。用血吸虫成虫冰冻切片以抗rSj29蛋白单克隆抗体的小鼠腹水作为一抗行间接免疫荧光实验观察2株单克隆抗体在日本血吸虫成虫的识别位点。用棋盘滴定法同时选择包被用抗rSj29兔多克隆抗体、抗rSj29小鼠单克隆抗体以及HRP-山羊抗小鼠抗体的工作浓度。结果成功克隆并用原核系统表达rSj29蛋白,纯化的rSj29膜外蛋白用Western blotting证实能够被急性、慢性和晚期日本血吸虫感染患者血清识别而与正常人血清无反应。免疫荧光定位结果证实了生物信息学的推测——Sj29蛋白是日本血吸虫成虫表膜蛋白,该蛋白在日本血吸虫成虫的体被表达非常丰富。rSj29-ELISA和AWA-ELISA两种方法平行检测急、慢性和晚期血吸虫病患者,华支睾吸虫患者,肠道线虫患者以及正常人血清抗体,结果为: rSj29-ELISA法检测急性、慢性和晚期日本血吸虫病患者血清抗体的敏感性分别为91.4%、89.5%和96.3%,与AWA-ELISA(91.4%、89.5%和88.9%)相比,两者敏感性统计学分析上无显著性差异;检测钩虫、华支睾吸虫感染者血清抗体的结果显示rSj29-ELISA(17.2%、8.1%)与AWA-ELISA(27.6%、18.5%)相比前者的交叉反应率绝对值较后者低,但统计学上无显著性差异,需加大样本量进一步确认;检测日本血吸虫感染治疗半年后患者血清抗体的阴转率两者相比有统计学显著性意义,前者(55.9%)的阴转率明显比后者(8.9%)的高,说明rSj29-ELISA可考虑作为疗效考核的重要参考指标之一。与此同时获得两株特异性分泌抗Sj29单克隆抗体的杂交瘤细胞株,经过体外多次传代培养后,仍能分泌高效价的抗体,细胞上清效价稳定在1:800~1:3200之间。用BALB/c小鼠制备单抗腹水,ELISA法测效价在1.024×105以上。两株杂交瘤细胞的抗体亚型均为IgG1。Western-Blotting证实两株细胞均为分泌抗Sj29单克隆抗体的杂交瘤细胞。免疫荧光实验结果显示两株杂交瘤细胞抗体的识别位点在日本血吸虫成虫的体被表膜上。初步建立了双抗体夹心ELISA用于血吸虫病人血清循环抗原检测的实验方法。结论本研究获得了预期的rSj29膜外区融合蛋白,证实了该重组蛋白具有较好的免疫反应性和免疫原性,间接免疫荧光结果显示Sj29是日本血吸虫成虫表膜蛋白。以rSj29膜外区融合蛋白包被建立了rSj29-ELISA方法用于血吸虫病人血清循环抗体的检测,获得较好的敏感性和特异性,尤其在疗效考核方面比AWA-ELISA更具优越性。该重组抗原制备简便,成本低廉,制备周期短,方法易于标准化,更适合商品化生产和流行区血吸虫病的血清学辅助诊断。同时获得了2株特异性分泌抗Sj29单克隆抗体的细胞株,且细胞上清和腹水中单克隆抗体的效价均较高。两株单抗亚型均为IgG1。Western-Blotting结果证实了该2株单克隆抗体的免疫学特性,免疫荧光实验结果证实两株杂交瘤细胞抗体的识别位点位于日本血吸虫成虫的体被表膜上。初步建立了双抗体夹心ELISA方法用于检测日本血吸虫病人血清中循环抗原。
[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
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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