霉菌毒素广谱型抗体的制备及其活性机理研究

Food and feed contamination by trichothecenes have caused serious health effect to human and animal, which are a group of over 180 structurally related sesquiterpenoid mycotoxins produced by Fusarium, Stachybotrys with structure in common. Antibody based immunoassay provide rapid test system for the Low molecular weight toxin determination, due to the Irreconcilable contradiction between the specificity character of antibody with limited cross-reactivity and broadly recognizing requirement,the antibody producing becomes more difficult and complicated, especially for a group of low molecular weight toxins determination. So it certainly needs to understand and profile the molecular interaction mechanism between antibody and antigen of low molecular weight, which will provide crucial information for the broadly recognizing antibody developing..With the advance of the crystal structure of the antibody-antigen complex study, only a limited number of complementarity determining regions (CDRs) amino acids residues actually participate in direct antigen-binding interactions, which have decisive roles for the antibody specificity, antibody character can be changed dramatically, if crucial amino acid mutated, the CDR loops form highly exposed domains that interact with antigen and these few amino acids located in CDR were clarified as the crux of docking to the antigen; So the hypothesis is: if the tip or pocket of the CDR of Single-chain variable fragment antibody(ScFv) rebuild with mutated, replaced or reduced crucial amino acid, which can insert into the receptor binding pocket on the mycotoxin common part, mimicking in many respects the interaction of the likeness physiological receptor, then the recombinant antibody broad reactivity to the mycotoxins of trichothecenes can be achieved by binding a particular conserved region, and also the interaction mechanism between binding sites of antibody and mycotoxin can be clarified with the relationship study between antibody 3D structure and its crucial amino acids docking sites. .Based on the hybridoma cell lines against deoxynivalenol (DON), which we already achieved and stored in Lab, and the B cell get from spleen of mice, which injected by antigen of DON-BSA and T2-BSA,Phage-display Library will be established for the variable fragment screening, and the variable anti-mycrotoxin antibody regions of heavy chain (VH) and light chain (VL) were arranged in VH-VL orientation, then expressed in Escherichia coli and insect cell line sf9, meanwhile x-ray and mimicking software will be employed for the mAb crystal structure study and Scfv antibody 3D structure analysis, and the possible crucial amino acids in the binding site will be identified, then with the profiled and better understood docking pocket, several ScFv antibodies will be chosen for the amino acid mutating, replacing and reducing aim at the broadly recognizing recombinant antibody developing and interaction mechanism analyzing.

霉菌毒素是食源性污染的重要毒素物质，结构相似种类繁多，具有很强的毒性。以抗体为基础的检测方法，对小分子的检测存在诸多瓶颈，特别是对一类结构相似的小分子毒素，抗体特异性与广谱性的统一仍是难点，确切的机制尚未阐明；课题以霉菌毒素为对象，拟通过改造抗体结合区（顶角区），使之能够识别一类毒素分子的保守区，以单克隆抗体细胞株及免疫的小鼠脾细胞为材料，建立噬菌体抗体库，进行基因筛选。然后比较分析单链抗体V区的空间构象，采用x射线及计算机模拟方法，分析抗体-毒素分子相互作用的立体构象、确定参与结合的关键氨基酸残基，研究活性氨基酸位点与结合构象的关系，采用氨基酸替换、突变、缩减等方法，改变CDR区的氨基酸组成，阐明广谱型基因工程抗体的活性机理，达到可识别家族类毒素分子的目的。课题采用重组抗体可变区基因、生物信息学模拟手段，构建表达系统，从重组基因的角度，为抗体特异性与广谱性的统一提供新思路。

本研究采用抗体基因工程技术、分子对接与模拟、噬菌体展示多肽库等技术手段，对广谱型抗体进行了基因工程制备与机理研究，获得了大量的用于基因工程改造材料，初步证实原设想可行，进展发现如下：（1）利用噬菌体展示技术建立了天然鼠源抗体库，已经初步筛选获得多个霉菌毒素(DON)抗体可变区基因，为后续抗体基因比较、分子对接与模拟，关键氨基酸突变提供了材料；（2）分子对接与模拟分析发现：DON抗体可变区关键结合位点的氨基酸若突变为疏水性更强的ILE（异亮氨酸）后，抗体与毒素的结合作用变强，若突变为ARG（精氨酸），抗体与毒素的结合变弱，为后续广谱型基因改造进行了有益的探索；（3）基于霉菌毒素玉米赤霉烯酮（ZEN）抗体对玉米赤霉醇（ZER）具有很高的交叉识别率，且两种化合物结构相似的特点，利用杂交瘤细胞技术获得了玉米赤霉醇（ZER）单克隆细胞株，并建立检测方法，为后续比较分子模拟提供了基础；（4）利用多肽库(PhD-12)技术对霉菌毒素的抗原表位的筛选，并建立了斑点快速检测方法，为后续的分子模拟对接比较分析奠定基础。上述结果初步证实并明确了利用人工改造抗体基因，结合分子模拟对接分子改造，可获得广谱型抗体并可作为机理研究的方法可行，并已获得多种研究材料。