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Updated: Apr 24, 2026
Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2
Published on: October 24, 2016
[Construction and application of a lentiviral vector of single-chain variable fragment antibody against human
Yonghua Chen1, Jia Guo2, Yanxin Yin2
1Department of Clinical Laboratory, Suzhou Municipal Hospital, Suzhou 215000, China.
Objective:
To construct a lentiviral expression vector carrying the single-chain variable fragment (scFv) antibody against human hepatocyte growth factor receptor (HGFR), express it in transfected HEK293 cells, and observe its biological function of specific binding to antigen.
Methods:
The variable regions of the heavy chain (VH) and light chain (VL) genes were amplified directly from the cDNA of hybridoma cell line 8E8 secreting mouse anti-human HGFR antibody and assembled using the splice overlap extension-PCR (SOE-PCR). The constructed HGFR-scFv gene with the signal peptide SP-VH-linker-VL was ligated into the cloning vector pCR-Blunt. After cut off from pCR-Blunt using enzyme digestion, HGFR-scFv gene was subcloned into the lentiviral transfer vector pRRL-CMV, which was identified by the restriction enzyme digestion and sequencing. The lentiviral expression vector pRRL HGFR-scFv was then tansfected together with the packaging plasmids into HEK293T cells to obtain virus particles, and green fluorescent protein (GFP) expression was detected under a fluorescent microscope. Then the virus particles were used to infect HEK293 cells. The scFv expression was detected by RT-PCR and its biological affinity as antibody was measured by ELISA.
Results:
The lentiviral expression vector pRRL HGFR-scFv was constructed correctly. After HEK293T cells were transfected with the pRRL HGFR-scFv plasmid, the GFP was visible. After HEK293 cells were infected with virus particles, the scFv antibody expressed could bind to HGFR specifically.
Conclusion:
The lentiviral expression vector of HGFR-scFv was constructed successfully, which would help to study the important role of HGFR in following experiments.
