摘要
目的研发靶向免疫球蛋白超家族成员9(IGSF9)的特异阻断型单克隆抗体,并验证该抗体的功能。方法将带有His标签的IGSF9细胞外结构域(IGSF9 extracellular domain,IGSF9-ECD)克隆到pcDNA3.1(-)的表达载体上,转染HEK293细胞,亲和层析获得IGSF9-ECD-His蛋白,应用SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western blotting进行蛋白鉴定。应用IGSF9-ECD-His蛋白免疫6周龄BALB/c小鼠4次,ELISA和流式细胞仪检测小鼠抗血清效价。分离小鼠脾脏细胞,与小鼠多发性骨髓瘤细胞SP2/0进行融合,ELISA和流式细胞仪筛选阳性克隆,并进一步筛选亚克隆细胞,制备靶向IGSF9的单克隆抗体。将IGSF9抗体加入到T细胞和THP-1细胞共培养体系中,检测上清中IFN-γ和TNF-α水平。结果IGSF9-ECD-His蛋白条带单一,分子量与预期相一致。在T细胞培养体系中加入该蛋白,显著抑制T细胞增殖。IGSF9-ECD-His蛋白免疫小鼠后,小鼠抗血清效价为1∶364500,而且抗血清能够显著逆转THP-1对T细胞的增殖抑制。本研究获取了64株克隆阳性细胞株,其中有6株能够用于流式细胞仪检测。本研究选择了阻断效应最好的5C3母克隆制备单克隆抗体,流式细胞仪和T细胞共培养结果筛选后,选择了阻断效应最好的5C3-1A4作为IGSF9单克隆抗体(anti-IGSF9),该抗体能够显著逆转IGSF9对T细胞的增殖抑制,并促进IFN-γ和TNF-α的分泌。结论本研究成功制备了IGSF9单克隆抗体,而且该抗体能够显著逆转IGSF9对T细胞增殖抑制。
Objective To develop specific blocking antibodies targeting immunoglobulin superfamily member 9(IGSF9),and observe the role of this antibody in tumor therapy.Methods The IGSF9 extracellular domain(IGSF9-ECD)with His tag was cloned into the expression vector pcDNA3.1(-),transfected into HEK293 cells,and IGSF9-ECD-His protein was obtained by affinity chromatography.Proteins were identified by SDS-Polyacrylamide gel electrophoresis(SDS-PAGE)and Western blotting.BALB/c mice aged 6 weeks were immunized with IGSF9-ECD-His protein.After 4 immunizations,mouse serum was separated and detected by ELISA and flow cytometry.Spleen cells were isolated and fused with mouse multiple myeloma cells SP2/0.Positive clones were screened by ELISA and flow cytometry,and subclones were further screened.Anti-IGSF9 was added into the T cell co-culture system with THP-1 cells.After 5 days,TNF-αand IFN-γin supernatants were detected by ELISA.Results SDS-PAGE and Western blotting results showed that IGSF9-ECD-His protein had a single band and the molecular weight was consistent with the expected,and could significantly inhibit T cell proliferation.After immunizing mice with IGSF9-ECD-His protein,the serum titer of mice was 1∶364500,and the serum could significantly reverse the inhibition of T cell proliferation by THP-1.Next,64 clonal positive cell lines were obtained,of which only 6 could be detected by flow cytometry.We selected 5 C3 with the best blocking effect to prepare monoclonal antibody and 5 C3-1 A4 was the monoclonal cell with the best blocking effect by T cell co-culture and flow cytometry.Therefore,we selected 5 C3-1 A4 cell to prepare monoclonal antibody targeting IGSF9.Anti-IGSF9(Clone No.5 C3-1 A4)significantly reversed the inhibition of T cell proliferation,and increased the levels of TNF-αand INF-γ.Conclusion Our group successfully prepared IGSF9 monoclonal antibody,which could significantly reverse the inhibition of T cell proliferation.
作者
刘祎璠
赵鑫钰
栾惠雯
刘晓丽
李尊岭
LIU Yifan;ZHAO Xinyu;LUAN Huiwen;LIU Xiaoli;LI Zunling(Department of Biochemistry and Molecular Biology,School of Basic Medicine,Shandong Tumor Immunotherapy Research Innovation Team,Binzhou Medical University,Yantai 264003,Shandong,P.R.China)
出处
《滨州医学院学报》
2022年第4期241-247,共7页
Journal of Binzhou Medical University
基金
国家自然科学基金(81872332,82070225,82150101)。
