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目的构建可表达抗人肝细胞生长因子受体(HGFR)单链抗体(scFv)的慢病毒表达载体,将其感染HEK293细胞,检测该抗体的表达及与抗原结合活性。方法采用反转录PCR(RT-PCR)从分泌小鼠抗人HGFR抗体的杂交瘤细胞株(8E8)中扩增VH和VL基因,用重叠延伸PCR法将VH和VL进行拼接,得到含有信号肽SP-VH-linker-VL的单链抗体基因(HGFR-scFv),将其插入克隆载体pCR-Blunt中。用内切酶将HGFR-scFv基因从pCR-Blunt上切下,插入慢病毒载体pRRL-CMV中,经相应酶切和测序鉴定,正确构建慢病毒表达载体pRRL HGFR-scFv。磷酸钙法转染HEK293T细胞,48 h后通过荧光显微镜检测绿色荧光蛋白(GFP)的表达,将获得病毒颗粒进一步感染HEK293细胞,RT-PCR和ELISA检测scFv的表达情况。结果抗人HGFRscFv的慢病毒表达载体构建成功,病毒颗粒感染HEK293细胞后,得到了可以稳定表达抗人HGFR-scFv的细胞系;ELISA结果表明,scFv能与人HGFR特异性结合。结论成功克隆了小鼠抗人HGFR抗体的scFv基因,并构建其慢病毒表达系统。
Objective To construct a lentiviral vector expressing human anti-human hepatocyte growth factor receptor (scFv) and infect HEK293 cells to detect the expression of the lentiviral vector and its binding activity with antigen. Methods VH and VL genes were amplified by reverse transcription-polymerase chain reaction (RT-PCR) from the hybridoma cell line secreting mouse anti-human HGFR antibody (8E8). The VH and VL genes were spliced by overlap extension PCR, The single-chain antibody gene (HGFR-scFv) of peptide SP-VH-linker-VL was inserted into the cloning vector pCR-Blunt. The HGFR-scFv gene was excised from pCR-Blunt by restriction endonuclease and inserted into the lentiviral vector pRRL-CMV. The lentiviral vector pRRL HGFR-scFv was constructed by restriction enzyme digestion and sequencing. HEK293T cells were transfected with calcium phosphate. The expression of green fluorescent protein (GFP) was detected by fluorescence microscopy 48 hours later. The virus particles were further infected HEK293 cells. The expression of scFv was detected by RT-PCR and ELISA. Results The lentiviral vector expressing human anti-human HGFRscFv was constructed successfully. The HEK293 cells were infected with virus particles, and the cell lines stably expressing anti-human HGFR-scFv were obtained. The results of ELISA showed that scFv could specifically bind to human HGFR. Conclusion The scFv gene of mouse anti-human HGFR antibody was successfully cloned and its lentiviral expression system was constructed.