噬菌体展示筛选人源细胞黏附分子L1结构域抗体及其体外实验研究

Screening of the domain antibody targeting cell adhesion molecule L1 through phage display and its in vitro effects

目的使用噬菌体展示技术筛选人源细胞黏附分子L1结构域抗体并体外观察其对神经来源细胞信号通路及细胞聚集的影响。方法以L1纤黏连蛋白FN2-3区肽段为抗原,利用噬菌体抗体库(DAb library)筛选并纯化相应人源结构域抗体,并通过酶联免疫吸附法(ELISA)及免疫荧光观察抗体与抗原结合特性。给予神经来源SK-N-SH细胞抗体,使用蛋白质印迹(Western blot)观察24 h时其对L1下游信号通路Erk及Akt1激活的影响。使用结晶紫进行细胞染色观察抗体对细胞聚集的影响。结果成功筛选出与L1FN2-3区肽段结合的结构域抗体H2-1,与SK-N-SH细胞内源性L1结合良好。与溶剂对照组相比,该抗体可有效促进SK-NSH细胞中L1下游信号分子Erk1/2及Akt1磷酸化激活(分别为P<0.01和P<0.05),并可促进神经突生长及神经细胞聚集。结论 L1激动型结构域抗体可激活L1相关信号分子,促进神经突生长等作用,具有进一步实验治疗脊髓损伤等神经系统创伤性疾病的研究价值。

Aims To screen the domain antibody tar- geting human cell adhesion molecule L1 through phage display, and to observe its effects on cell signaling pathways and cell accumulation. Methods With the fibronectin type Ⅲ repeats Ⅱ- Ⅲ domain （FN2-3） de- rived from human cell adhesion molecule L1 as the an- tigen, phage domain antibody library （DAb library） was used to identify- its colwesponding antibody. En- zyme-linked inmmnosorbent assay （ELISA） and inmmnofluorescence staining were employed to identify- its affinity to the recombinant antigen and intrinsic L1. Human neuroblastoma SK-N-SH cells were treated with the domain antibody for 24 h. Its effects on Ll-related cell signaling pathways were evaluated by Western blot, and the change of cell accumulation was investi- gated by crystal violet staining. Results A domain antibody （H2-1） was successfully screened to target FN2-3 domain and could bind to the intrinsic cell ad- hesion molecule L1 localized on the SK-N-SH cells. Compared to the vehicle control group, administration d domain antibody H2-1 significantly increased the for- marion d phosphorylated ERK1/2 （pERK1/2） and phophorylated Aktl （pAktl） （P 〈 0.01 and P 〈0. 05, respectively）. Cell accumulation was also ob- served in response to these effects. Conclusion Do- main antibody targeting L1 can activate ERK1/2 and Aktl, and induce the prolongation of cellular proces- ses, which may wan＇ant the experimentally therapeutic research for neuronal injury diseases such as spinal cord injury.