摘要
黄曲霉菌(Aspergillus flavus)是一种重要的植物病原真菌,造成玉米、花生、棉花等多种农作物的减产和粮食的霉变,导致重大的经济损失。黄曲霉菌也是一种重要的人和动物致病菌引起严重的曲霉病。黄曲霉菌和寄生曲霉菌(Aspergillus parasiticus)产生的次级代谢产物黄曲霉毒素(aflatoxin)是一种剧毒的真菌毒素,具有强烈地致癌和致畸作用。被黄曲霉菌污染的农产品中通常有大量的黄曲霉毒素残留,被人和动物误食会导致严重的中毒事件,长期摄入会诱发肝癌等疾病,严重威胁人和动物的健康。因此对黄曲霉菌进行快速、灵敏地检测对于预防黄曲霉菌引起的动植物病害,监测黄曲霉毒素的污染具有重要的意义。
免疫学检测技术利用了抗体和抗原之间的特异性互作,已经被广泛应用于各种病原菌的快速诊断。传统的免疫学检测方法主要利用多克隆抗体和单克隆抗体,但这2种抗体的制备依赖于动物免疫和杂交瘤细胞融合技术,生产成本较高。利用噬菌体展示技术可以从抗体文库中筛选到高亲和力单链抗体,并且同时获得抗体的编码基因。单链抗体具有易于原核表达和基因工程改造等优点,在免疫学检测领域中有显著的优势。本研究以黄曲霉菌细胞壁蛋白为靶标,选到了高亲和力单链抗体,所取得的主要成果如下：
1.将黄曲霉细胞壁蛋白免疫小鼠,通过杂交瘤细胞融合技术筛选到了4株黄曲霉菌特异单克隆抗体杂交瘤细胞。对4种从小鼠腹水中纯化的单克隆抗体进行了ELISA分析,结果表明rnAb2A8抗体对黄曲霉细胞壁蛋白的亲和力最高,可以用于免疫学检测。
2.将黄曲霉细胞壁蛋白免疫鸡,构建了库容量为1.2×107的鸡源单链抗体文库,并利用噬菌体展示技术对抗体文库进行了筛选。通过表达ELISA从筛选后的单链抗体文库中鉴定出了35个阳性克隆,对所属的4种类型高亲和力抗体AfSA4、AfSA5、 AfSA8和AfSD10进行原核表达和纯化。
3.对筛选到的单链抗体氨基酸序列进行分析,结果表明抗体CDR区序列的组成和长度存在较大差异。间接ELISA分析表明AfSA4对黄曲霉菌和寄生曲霉菌的亲和力最高。对AfSA4、AfSA5、AfSA8和AfSD10的空间结构进行了预测,结构比较分析表明AfSA4的L-CDR2和H-CDR3结构域之间存在氢键等紧密的相互作用,该部位独特的致密空间结构可能与AfSA4的高亲和力密切相关。免疫荧光分析表明AfSA4可以与黄曲霉和寄生曲霉细胞壁表面结合,而AfSA5、AfSA8和AfSD10不能识别细胞壁表面。免疫印迹分析也进一步证明了不同单链抗体之间存在抗原识别特性的差异。
4.利用phage ELISA方法对淘选后的抗体文库进行鉴定,对筛选到的阳性克隆序列进行了分析,并与表达ELISA的鉴定结果进行比较。从中筛选到了高亲和力单链抗体AfPD12,并进行原核表达和纯化。免疫荧光实验证明AfPD12识别黄曲霉和寄生曲霉细胞壁表面抗原。免疫印迹实验证明AfPD12识别细胞壁蛋白。
5.将AfSA4和AfPD12与碱性磷酸酶(AP)构建成融合蛋白AfSA4-AP和AfPD12-AP,并进行原核表达和纯化。ELISA和免疫印迹分析表明scFv抗体和scFv-AP融合蛋白具有相同抗原识别特性,特异性识别曲霉属病原真菌。表面等离子共振(SPR)分析表明AfSA4-AP亲和力比AfSA4提高了约6倍,AfPD12-AP亲和力比AfPD12提高了约14倍。间接ELISA实验表明scFv-AP融合蛋白检测抗原的灵敏度更高。
6.将AfPD12与生物素化标签蛋白构建了Bio-AfPD12融合蛋白,并进行原核表达和纯化。Western blot分析表明链霉亲和素特异性识别原核表达的Bio-AfPD12证明Bio-AfPD12被成功生物素化。间接ELISA实验表明Bio-AfPD12能够用于检测黄曲霉菌和寄生曲霉菌。
7.利用包被抗体mAb2A8和检测抗体AfSA4-AP建立了夹心ELISA(ds-ELISA)检测体系,可以同时对黄曲霉菌和寄生曲霉菌进行高灵敏度检测,对两者的最低检测限达到10q-3μg/mL。在玉米和花生基质中对两种真菌检测的灵敏度达到1μg/g。该检测方法对健康的玉米、花生材料和非曲霉属的常见植物病原真菌没有交叉反应,可以用于对黄曲霉毒素产生菌的免疫学检测。
8.利用包被抗体AfPD12和检测抗体AfPD12-AP建立的ds-ELISA可以检测黄曲霉菌和寄生曲霉菌,灵敏度达到10-2μg/mL,该方法可以避免对单克隆抗体的使用。利用AfPD12和Bio-AfPD12建立了夹心荧光免疫吸附(ds-FLISA)检测方法,对黄曲霉菌和寄生曲霉菌的检测灵敏度为10-1μg/mL
综上所述,本研究利用噬菌体展示技术筛选得到了多种类型黄曲霉菌特异单链抗体,并从空间结构和免疫识别等多个方面对抗体的特性进行了分析。将高亲和力单链抗体构建了scFv-AP和Bio-scFv融合蛋白,获得了双功能抗体融合蛋白。将单克隆抗体、单链抗体及其融合蛋白相互结合建立了ds-ELISA和ds-FLISA检测方法。本研究建立的免疫学检测方法可以对黄曲霉毒素产生菌进行有效检测,对保障农作物生产和食品生产安全具有重要的意义。
The Aspergillus flavus is important plant pathogen that infects agricultural crops before harvest and during storage. Usually, A. flavus usually infects maize, peanuts and cotton causing crop yield loss, food mildew and huge economic losses. Additionally, A. flavus is also a human and animal pathogen, which leads to serious aspergillosis disease. A. flavus and A. parasiticus are the predominant producers of aflatoxins, which are secondary metabolites with carcinogenic and mutagenic effects to human and animals. The infection of A. flavus in agricultural products usually leads to high-level aflatoxin contamination, which is a severe threaten to human and animal health. High concentrations of aflatoxins intake will cause acute aflatoxicosis, and daily intake of low dose of aflatoxins will induce liver cancer and other diseases. Thus detection of A. flavus fast and sensitively will play a critial in prevention of disease caused by A. flavus and monitoring the contamination of aflatoxins.
Immunoassay has been widely used in the detection of various pathogens. Immunoassay is based on the specific recognition between antigen and antibody, so antibody with high affinity palys an essential role in this system. Traditional immunoassay relies on polyclonal and monoclonal antibody, which are produced by animal immunization and hybridoma cell technology, and the cost of production is usually high. High-affinity single-chain antibody (scFv) can be isolated from immune or naive antibody library through phage display. In addition, phage display selection system can immediately provide gene sequences of single-chain antibodies against the target. The single-chain antibody can be easily expressed in bacteria and fused to other moleculars by subcloning which have advantages in immunoassay. In this study, we aimed to isolate single-chain antibody against A. flavus cell wall target from an immunized chicken antibody library using phage display. Based on scFv fusion proteins, new kinds of Aspergillus pathogens immunoassay methods were developed. The main results of this study are as follows:
1. A.flavus cell wall proteins were used as antigen to immunize Balb/c mice. Four A. flavus specific monoclonal antibodies were isolated through hybridoma cell technology and purified from ascitic fluids. Among them mAb2A8showed the strongest binding ability in indirect ELISA, and can be used in immunoassay.
2. A. flavus cell wall proteins were used as antigen to immunize White Leghorn chickens. The genes of variable domians of heavy chain and light chain were cloned and constructed into a recombinant antibody library with a size of1.2×107. The antibody library was screened using phage display, and35positive clones were identified through expression ELISA. There were four types of antibodies named AfSA4, AfSA5, AfSA8and AfSD10according to their amino acid sequences. These scFv antibodies were expressed in bacteria and then purified using affinity chromatography.
3. Alignment of amino acid sequences from AfSA4, AfSA5, AfSA8and AfSD10showed that there is variation in the composition of amino acid and length of CDR regions. Indirect ELISA showed that AfSA4exhibited the strongest binding activity towards A. flavus and A. parasiticus. The3D model prediction of scFv antibodies were also performed, and the comparison of structures showed that the interaction (e.g. hydrogen bond) between the loops of L-CDR2and H-CDR3of AfSA4facilitates the forming of a compact motif, which may play a critical role in high binding ability. Immunofluorescence showed that the binding of AfSA4localized to the cell wall of A. flavus and A. parasiticus. Whereas, antibodise AfSA5, AfSA8and AfSD10were not able to bind to the surface of cell wall. Immunoblot analysis showed that the binding properties of different scFv antibodies are varied.
4. Phage ELISA was also used to identify positive clones from library after panning. The sequences of positive clones were compared with those obtained by expression ELISA. A high reactive scFv antibody AfPD12was expressed in bacteria and purified consequently. Immunofluorescence showed that AfPD12also binds to the cell wall of A. flavus and A. parasiticus. Immunoblot demonstrated that AfPD12recognizes the cell wall proteins of A. flavus.
5. The genes of AfSA4and AfPD12were fused to that of alkaline phosphatase (AP) to generate fusion protein AfSA4-AP and AfPD12-AP, which were expressed and purified in bacteria. ELISA and immunoblot showed that scFv and scFv-AP exhibited the same specificity towards Aspergillus, so AP did not interfere the binding property of scFv in this study. The simultaneous dimerization of AP molecular imparts avidity to scFv-AP, which would improve the binding performance of scFv-AP. Surface plasmon resonance (SPR) analysis showed that the affinity of AfSA4-AP fusion was about6-fold higher than AfSA4, and AfPD12-AP also exhibited14-fold higher affinity compared with AfPD12. Indirect ELISA demonstrated that scFv-AP fusions were more sensitive in immunoassay.
6. The gene of AfPD12was fused to a protein tag that can be biotinylated when expressed in bacteria. The bio-AfPD12fusion protein was expressed in bacteria and purified consequently. Western blot analysis showed that bio-AfPD12was successfully biotinylated, and streptavidin specifically recognized bio-AfPD12. Indirect ELISA showed that bio-AfPD12could be used in the immunoassay.
7. Monoclonal antibody mAb2A8was used as capture antibody, and AfSA4-AP fusion was used as detection antibody to set up a sensitive sandwich ELISA (ds-ELISA). This ds-ELISA was able to detect A. flavus and A. parasiticus simultaneously with a detection limit of10-3μg/mL. The sensitivity reached up to1μg/g when the ds-ELISA was used to detect fungi in maize and peanut matrix. The ds-ELISA had no cross reactivity towards health materials and non-Aspergillus fungi, so this immuassay can be used in the detection of aflatoxin producing fungi.
8. Capture antibody AfPD12and detection antibody AfSA4-AP fusion were used to set up a sandwich ELISA, which showed a sensitivity of10-2μg/mL. The using of capture antibody AfPD12avoided the need of monoclonal antibody. A double sandwich fluorescence-linked immunosorbent assay (ds-FLISA) was developed based on AfPD12and bio-AfPD12. The detection limits of ds-FLISA against A. flavus and A. parasiticus were10-1μg/mL.
In summary, different scFv antibodies were isolated from chicken antibody library using phage display, and their properties were analyzed through structure and immunorecognition in this study. ScFv antibodies with higher affinity were constructed into biofunctional scFv-AP and bio-scFv fusion. The well-matched monoclonal antibody, scFv and its fusion proteins were used to develope ds-ELISA and ds-FLISA method. The immunoassay developed in this study can be used in the detection of aflatoxin producing fungi, and will play an important role in ensuring agricultural production and food safety.
引文
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