4-戊基苯酚单克隆抗体的制备及其可变区的同源建模

Preparation of monoclonal antibody against 4-amylphenol and homology modeling of its Fv fragment

目的制备4-戊基苯酚(4-AP)单克隆抗体(mAb),通过分子生物学方法对其可变区cDNA克隆和同源建模,为4-AP单链抗体制备及抗体分子层面改造奠定基础,同时为4-AP的免疫学检测提供一种新方法。方法将免疫小鼠脾脏细胞与Sp2/0细胞融合,筛选出F10阳性单抗细胞株。通过其mRNA的抽提,克隆出可变区cDNA序列,利用生物软件进行同源建模和分子对接。结果在最适条件下,其间接竞争ELISA(ic-ELISA)公式为y=A_2+(A_1-A_2)/(1+(x/x_0)~p)(A_1=1.28;A_2=-0.07;x_0=12 560.75;p=0.74),相关系数(R^2)为0.997,其最低检测限为0.65μg/mL。mAb重链和轻链分别属于IgG1型和Kappa型,其与4-AP的结合力为氢键和疏水作用。结论成功制备出1株稳定分泌的4-AP mAb的杂交瘤细胞株。通过同源建模对抗体可变区空间结构进行深入研究,为单链抗体及抗体改造提供参考,同时为4-AP含量检测提供新方法。

Objective To prepare and characterize a monoclonal antibody（mAb） against 4-amylphenol（4-AP）,clone its cDNA sequence and make homology modeling for its Fv fragment. Methods A high-affinity anti-4-AP mAb was generated from a hybridoma cel line F10 using electrofusion between splenocytes from APA-BSA-immunized mouse and Sp2/0 myeloma cells. Then we extracted the mRNA of F10 cells and cloned the cDNA of mAb. The homology modeling and molecular docking of its Fv fragment was conducted with biological software. Results Under the optimum conditions,the ic-ELISA equation was y = A2＋（A1-A2）/（1 ＋（x/x0）-p）（A1= 1. 28; A2=-0. 066; x0= 12 560. 75; p = 0. 74） with a correlation coefficient（R-2） of 0. 997. The lowest detectable limit was 0. 65 μg/mL. The heavy and light chains of mAb respectively belonged to IgG1 and Kappa. The homology modeling and molecular docking studies revealed that the binding of 4-Ap and mAb was attributed to the hydrogen bond and hydrophobic interactions. Conclusion The study successfully established a stable 4-AP mAb-secreting hybridoma cel line. The study on spatial structure of Fv fragment using homology modeling provided a reference for the development and design of single chain variable fragments.