日本血吸虫感染血清中循环抗原的筛选与鉴定
发布时间：2018-07-13 10:52
【摘要】：寻找优势诊断抗原,一直是血吸虫病诊断研究中的热点。血吸虫循环抗原因其具有反映活动性感染情况、评估虫负荷和疗效考核价值等特点,一直倍受人们关注,寻找高敏感性和高特异性方法检测循环抗原,也是血吸虫病诊断研究长期面临的挑战。 鉴于本研究组在基因工程抗体方面的前期研究工作,本课题旨在通过已经获得的SIEA26-28kDa SjscFv,利用其对日本血吸虫成虫、虫卵各发育阶段的高特异性靶向免疫识别能力,充分发挥其作为探针的作用,从日本血吸虫感染兔血清中,筛选血吸虫循环抗原组分,并利用NanoLC-MS/MS质谱以及生物信息学技术进行初步分析,鉴定循环抗原的相关组分,以期寻找具有高效诊断价值的血吸虫循环抗原,为建立高敏感性和高特异性检测循环抗原方法奠定基础。 研究目的 1.构建原核质粒pET43.1a/SjscFv,表达、纯化与鉴定SjscFv目的蛋白。 2.利用SjscFv筛选日本血吸虫感染兔血清中的循环抗原。 3.通过质谱和生物信息学技术手段,分析循环抗原相关组分的结构与功能,鉴定出具有诊断价值的候选抗原分子。 研究方法 1.原核质粒的构建及SjscFv蛋白的表达、纯化与鉴定 根据SjscFv基因开放阅读框架(ORF)设计上下游引物,以pET28a/SIEA26-28kDa-SjscFv质粒为模板,构建pET43.1a/SjscFv原核质粒,经PCR、双酶切和DNA测序鉴定重组质粒构建是否成功。以最佳诱导条件大量培养BL21(DE3)大肠杆菌表达SjscFv目的蛋白,Western blot鉴定该蛋白活性,Ni-柱层析法纯化蛋白并测定蛋白浓度。 2.日本血吸虫感染兔血清中循环抗原的筛选 通过SDS-PAGE和Native-PAGE凝胶电泳、Western Blot技术,利用pET43.1a/SjscFv蛋白识别日本血吸虫感染兔血清中的循环抗原相关组分,寻找感染阳性兔血清与重组蛋白特异结合的抗原条带。通过电泳收集特异条带并进行电洗脱纯化。同时,将纯化抗原与佐剂等体积混匀后免疫新西兰兔,制备循环抗原相关组分免疫血清,采用血吸虫成虫和感染兔肝制作石蜡切片,进行免疫荧光组化定位。 3.NanoLC-ESI-MS/MS质谱技术分析SjscFv识别的血吸虫感染兔血清中循环抗原的组分；通过生物信息学技术分析循环抗原相关组分的结构与功能。 研究结果 1.成功构建了原核质粒pET43.1a/SjscFv,经PCR、双酶切及DNA测序结果均证实所构建质粒pET43.1a/SjscFv中基因片段与目的片段一致。诱导表达pET43.1a/SjscFv工程菌,在约91kDa处获得大量表达蛋白,优化的最佳表达条件为：IPTG浓度为0.05mmol/L,温度为28℃,时间为过夜。采用Western Blot分析证实表达蛋白与目的蛋白一致。经镍柱层析法纯化后,SIEA26-28kDa-SjscFv蛋白浓度可达到0.339mg/mL。 2.采用SIEA26-28kDa-SjscFv进行Western Blot分析,发现SIEA26-28kDa-SjscFv可识别日本血吸虫感染2周-10周的阳性兔血清中的95KDa循环抗原相关组分。通过Native-PAGE证实该分子量约为95kDa。Western Blot和免疫荧光定位显示该循环抗原相关组分来源于成虫体表,而在虫卵中未出现,提示其与血吸虫感染早期的阶段性抗原相关。 3.通过SDS-PAGE电泳获得的循环抗原反应条带,经切胶电洗脱分离纯化后,采用NanoLC-ESI-MS/MS质谱技术进行分析,共鉴定出六种与血吸虫循环抗原相关的组分分别为：heat shock protein, actin, Importinβ1,Na+/K+transporting ATPase subunit alpha,SjCHGC04905protein,SjCHGC06108protein。Importin-β1作生物信息学分析,推测它主要具有HEAT repeat结构域、IBB-N结构域和ARM repeat结构域,α螺旋折叠成的三级结构可伸展或收缩,无跨膜区,存在丰富的抗原表位,预测其为较好的诊断候选分子。 结论 1.成功构建原核重组质粒pET43.1a/SjscFv并大量表达和纯化SjscFv重组蛋白； 2.筛选与鉴定出六种与血吸虫循环抗原相关的组分,该抗原在日本血吸虫成虫体壁大量表达,推测其与血吸虫感染早期的阶段性抗原相关； 3.Importin-β1蛋白的结构与功能分析推测它是一良好的诊断候选抗原分子。
[Abstract]:The search for superiority diagnostic antigen has been a hot spot in the diagnosis and research of schistosomiasis . It has been paid more attention to the diagnosis of schistosomiasis because it has the characteristics of reflecting active infection , evaluating the value of insect load and curative effect , and finding high sensitivity and high specificity method to detect circulating antigen . It is also a long - term challenge for the diagnosis of schistosomiasis .
In view of the previous research work on genetic engineering antibody , the aim of this study was to use the SIEA26 - 28kDa SjscFv which has been obtained to target the high - specific target immune recognition ability of Schistosoma japonicum adult and egg development stage , and to use the NanoLC - MS / MS mass spectrometry and bioinformatics technology to analyze the circulating antigen of Schistosoma japonicum , and to identify the relevant components of circulating antigen , so as to find the circulating antigen of Schistosoma japonicum with high efficiency diagnostic value , and lay the foundation for the establishment of high sensitivity and high specificity detection cycle antigen method .
Purpose of study
1 . Construction of prokaryotic plasmid pET43.1a / SjscFv , expression , purification and identification of SjscFv target protein .
2 . Using SjscFv to screen the circulating antigen of Schistosoma japonicum infected rabbit serum .
3 . By means of mass spectrometry and bioinformatics , the structure and function of circulating antigen - related components were analyzed , and the candidate antigen molecules with diagnostic value were identified .
Research Methods
1 . Construction of prokaryotic plasmid and expression , purification and identification of SjscFv protein
The prokaryotic plasmid pET43.1a / SjscFv was constructed by using pET28a / SIEA26 - 28kDa - SjscFv plasmid as template and pET28a / SIEA26 - 28kDa - SjscFv plasmid as template .
2 . Screening of circulating antigens in sera from rabbits infected with Schistosoma japonicum
By SDS - PAGE and Native - PAGE gel electrophoresis , Western Blot was used to identify the circulating antigen - related components of Schistosoma japonicum infected rabbit serum by using pET43 . 1a / SjscFv protein . The specific band was collected by electrophoresis and electroelution was carried out . At the same time , purified antigen was mixed with adjuvant and purified by electroelution .
3 . NanoLC - ESI - MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS /
The structure and function of circulating antigen - related components were analyzed by bioinformatics .
Results of the study
1 . The prokaryotic plasmid pET43.1a / SjscFv was successfully constructed . The recombinant plasmid pET43.1a / SjscFv was constructed by PCR , double digestion and DNA sequencing . The recombinant plasmid pET43.1a / SjscFv was constructed . The expression of pET43 . 1a / SjscFv engineering bacteria was induced . The optimal expression conditions were as follows : IPTG concentration was 0.05 mmol / L , temperature was 28 鈩，
本文编号：2119109
[Abstract]:The search for superiority diagnostic antigen has been a hot spot in the diagnosis and research of schistosomiasis . It has been paid more attention to the diagnosis of schistosomiasis because it has the characteristics of reflecting active infection , evaluating the value of insect load and curative effect , and finding high sensitivity and high specificity method to detect circulating antigen . It is also a long - term challenge for the diagnosis of schistosomiasis .
In view of the previous research work on genetic engineering antibody , the aim of this study was to use the SIEA26 - 28kDa SjscFv which has been obtained to target the high - specific target immune recognition ability of Schistosoma japonicum adult and egg development stage , and to use the NanoLC - MS / MS mass spectrometry and bioinformatics technology to analyze the circulating antigen of Schistosoma japonicum , and to identify the relevant components of circulating antigen , so as to find the circulating antigen of Schistosoma japonicum with high efficiency diagnostic value , and lay the foundation for the establishment of high sensitivity and high specificity detection cycle antigen method .
Purpose of study
1 . Construction of prokaryotic plasmid pET43.1a / SjscFv , expression , purification and identification of SjscFv target protein .
2 . Using SjscFv to screen the circulating antigen of Schistosoma japonicum infected rabbit serum .
3 . By means of mass spectrometry and bioinformatics , the structure and function of circulating antigen - related components were analyzed , and the candidate antigen molecules with diagnostic value were identified .
Research Methods
1 . Construction of prokaryotic plasmid and expression , purification and identification of SjscFv protein
The prokaryotic plasmid pET43.1a / SjscFv was constructed by using pET28a / SIEA26 - 28kDa - SjscFv plasmid as template and pET28a / SIEA26 - 28kDa - SjscFv plasmid as template .
2 . Screening of circulating antigens in sera from rabbits infected with Schistosoma japonicum
By SDS - PAGE and Native - PAGE gel electrophoresis , Western Blot was used to identify the circulating antigen - related components of Schistosoma japonicum infected rabbit serum by using pET43 . 1a / SjscFv protein . The specific band was collected by electrophoresis and electroelution was carried out . At the same time , purified antigen was mixed with adjuvant and purified by electroelution .
3 . NanoLC - ESI - MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS /
The structure and function of circulating antigen - related components were analyzed by bioinformatics .
Results of the study
1 . The prokaryotic plasmid pET43.1a / SjscFv was successfully constructed . The recombinant plasmid pET43.1a / SjscFv was constructed by PCR , double digestion and DNA sequencing . The recombinant plasmid pET43.1a / SjscFv was constructed . The expression of pET43 . 1a / SjscFv engineering bacteria was induced . The optimal expression conditions were as follows : IPTG concentration was 0.05 mmol / L , temperature was 28 鈩，
本文编号：2119109
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