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目的克隆大鼠抗小鼠的抗CD40激活型抗体的单链抗体(CD40-scFv),并构建可表达大鼠抗小鼠的抗CD40抗体的单链抗体(CD40-scFv)的重组慢病毒。方法采用RT-PCR法从分泌大鼠抗小鼠抗CD40激活型抗体的杂交瘤细胞株(FGK-115)中克隆VH和VL基因,用重叠延伸PCR法将VH和VL拼接在一起,构建抗CD40抗体的scFv基因,将CD40-scFv基因连接至PEGM-T克隆载体,限制性内切酶酶切及测序鉴定;构建含有大鼠抗小鼠的抗CD40激活型抗体的单链抗体(CD40-scFv)的慢病毒穿梭质粒,并与其它包装质粒一起通过磷酸钙法感染293T细胞;获得病毒颗粒并感染人慢性髓系白血病细胞MEG-01,检测其感染效率。结果含大鼠抗小鼠的抗CD40激活型抗体的单链抗体(CD40-scFv)的慢病毒穿梭质粒构建成功;通过磷酸钙感染293T细胞所得病毒上清可成功感染MEG-01细胞,流式细胞术检测阳性率可达96%。结论成功克隆了大鼠抗小鼠的抗CD40激活型抗体的单链抗体(CD40-scFv)基因,并建立其重组慢病毒表达系统,为后续的实验及应用研究工作奠定了基础。
Objective To clone a single chain antibody (CD40-scFv) of rat anti-mouse anti-CD40 activating antibody and construct a recombinant lentivirus which can express single chain antibody (CD40-scFv) of rat anti-mouse anti-CD40 antibody. Methods VH and VL genes were cloned from hybridoma cell line (FGK-115) secreting rat anti-mouse anti-CD40 active antibody by RT-PCR. VH and VL were spliced together by overlap extension PCR to construct anti-CD40 CD40 antibody scFv gene, the CD40-scFv gene was linked to the PEGM-T cloning vector, restriction endonuclease digestion and sequencing identification; construction of rat anti-mouse anti-CD40 activating antibody single chain antibody (CD40- scFv) lentiviral shuttle plasmids and infecting 293T cells by calcium phosphate method together with other packaging plasmids; obtaining virus particles and infecting human chronic myeloid leukemia cells MEG-01 to detect the infection efficiency. Results The lentiviral shuttle plasmid containing rat anti-mouse anti-CD40 activating antibody (CD40-scFv) was successfully constructed. The virus supernatant from 293T cells infected with calcium phosphate could successfully infect MEG-01 cells, Cytometry test positive rate up to 96%. Conclusion The single-chain antibody (CD40-scFv) of anti-mouse anti-CD40 antibody was successfully cloned and the recombinant lentivirus expression system was established, which lays the foundation for further experimental and applied research.