抗人肝细胞生长因子受体单链抗体慢病毒载体的构建和应用

发布时间：2018-05-12 00:00

本文选题：肝细胞生长因子受体 + 单链抗体　；参考：《细胞与分子免疫学杂志》2014年09期

【摘要】：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。
[Abstract]:Objective to construct a lentivirus expression vector which can express anti-human hepatocyte growth factor receptor (HGFR) single-chain antibody (scFv), and to detect its expression and antigen-binding activity in HEK293 cells. Methods VH and VL genes were amplified by reverse transcription PCR RT PCR from hybridoma cell line 8 E8 which secreted anti-human HGFR antibody. VH and VL were spliced by overlapping extension PCR. A single chain antibody gene (HGFR-scFv1) containing signal peptide SP-VH-linker-VL was obtained and inserted into the cloned vector pCR-Blunt. HGFR-scFv gene was digested from pCR-Blunt by endonuclease and inserted into lentivirus vector pRRL-CMV. The lentivirus expression vector pRRL HGFR-scFv was correctly constructed by restriction endonuclease digestion and sequencing. After transfection of HEK293T cells with calcium phosphate for 48 h, the expression of green fluorescent protein (GFP) was detected by fluorescence microscope. The viral particles were further infected with HEK293 cells by RT-PCR and ELISA to detect the expression of scFv. Results the lentivirus expression vector against human HGFRscFv was successfully constructed. After the virus particles were infected with HEK293 cells, a cell line capable of stably expressing anti-human HGFR-scFv was obtained by Elisa. The results showed that SSCFv could specifically bind to human HGFR. Conclusion the scFv gene of mouse anti-human HGFR antibody was cloned successfully and its lentivirus expression system was constructed.
【作者单位】：苏州市立医院东区检验科;同济大学苏州研究院生物医药中心;
【基金】：江苏省自然科学基金(BK2012162) 苏州市科技发展计划(SYG201131) 苏州市生物新药创制重点实验室(SZS201002)
【分类号】：R392.11

【相似文献】