构建CLDN18.2慢病毒载体及其稳转细胞株的建立

Establishment of CLDN18.2 lentiviral vector and its stable transformed cell line

目的构建表达人紧密连接蛋白18.2(CLDN18.2)的慢病毒载体,并通过慢病毒转导293T细胞获得稳定表达CLDN18.2的293T稳转细胞株。方法采用聚合酶链式反应(PCR)扩增目的基因,将其插入质粒pWPT,构建pWPT-CLDN18.2慢病毒包装质粒。采用PCR对重组质粒进行鉴定,对重组质粒进行慢病毒包装后转导293T细胞系,同时设立未转让质粒的293T细胞作为对照组,得到293T-CLDN18.2混合克隆细胞。检测转染后目的蛋白水平及单克隆细胞阳性率。结果经PCR鉴定,在700 bp处有一条清晰条带与目的基因大小相符。在25~35 kDa处有明显条带,与预期28.8 kDa相符。表达CLDN18.2单克隆细胞的阳性率为99.3%。结论该研究成功获得稳定表达人CLDN18.2的293T稳转细胞株,为下一步的单克隆抗体的制备奠定了基础。

Objective To construct a lentiviral vector expressing human tight junction protein 18.2(CLDN18.2),and to obtain a stable 293T transformed cell line expressing CLDN18.2 through lentiviral transduction of 293T cells.Methods The target gene was amplified by polymerase chain reaction(PCR)and inserted into the plasmid pWPT to construct the pWPT-CLDN18.2 lentivirus packaging plasmid.The recombinant plasmid was identified by PCR,and the recombinant plasmid was packaged with lentivirus and transduced into 293T cell line.At the same time,293T cells without transferred plasmid were set up as the control group to obtain 293T-CLDN18.2 mixed clone cells.The target protein level and monoclonal cell positivity rate after transfection were detected.Results A clear band at 700 bp was identified by PCR,that matches the size of the target gene.There was a clear band at 25-35 kDa,which was consistent with the expected 28.8 kDa.The positive rate of monoclonal cells expressing CLDN18.2 was 99.3%.Conclusion This study successfully obtains a stable 293T transgenic cell line expressing human CLDN18.2,and lays the foundation for the preparation of monoclonal antibodies in the next step.