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为构建抗黄曲霉毒素M1单链抗体(scFv)基因,并进行蛋白质结构模拟及理化特性分析。采用重叠延伸PCR方法,以能特异性分泌抗黄曲霉毒素M1单克隆抗体的杂交瘤细胞株1B12为原料,构建其单链抗体基因,进行测序,采用生物信息软件进行氨基酸序列推导、结构预测以及理化性质分析。抗黄曲霉毒素M1scFv基因全长737 bp,对应245个氨基酸,单链抗体分子量约为25 897.4,等电点预测值6.90;二级结构显示抗黄曲霉毒素M1单链抗体含α螺旋38处,β折叠24处,延伸链74处,随机卷曲109处。三级结构建模显示重链(VH)和轻链(VL)的6个环区(CDR区),共同组成抗体的抗原结合区,符合scFv的结构特点,具有抗原结合位点的空间构象。构建了一个抗黄曲霉毒素M1scFv基因,应用生物信息学技术所获得的预测和分析结果为该单链抗体的进一步表达、纯化和活性研究提供信息。
To construct aflatoxin M1 single chain antibody (scFv) gene, and protein structure simulation and physicochemical properties analysis. The overlapped extension PCR method was used to construct the single-chain antibody gene of hybridoma cell line 1B12 which can specifically secrete anti-aflatoxin M1 monoclonal antibody for sequencing. Bioinformatics software was used to deduce the amino acid sequence, structure prediction and Physical and chemical properties analysis. The anti-aflatoxin M1scFv gene was 737 bp in length, corresponding to 245 amino acids. The molecular weight of the single-chain antibody was 25 897.4 and the predicted isoelectric point was 6.90. The secondary structure showed that the aflatoxin M1 single chain antibody contained 38 α-helices, The beta sheet is folded at 24, extended at 74, and randomly curled at 109. The tertiary structure modeling showed that the six loop regions (CDR regions) of the heavy chain (VH) and the light chain (VL) together form the antigen binding region of the antibody and conformed to the structural features of the scFv with the spatial conformation of the antigen binding site. An aflatoxin M1scFv gene was constructed and predicted and analyzed using bioinformatics techniques to provide further information on the further expression, purification and activity studies of this single chain antibody.