title: [Construction and application of a lentiviral vector of single-chain variable fragment antibody against human hepatocyte growth factor receptor].
abstract: 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.
  
  
  CONCLUSIONS
  The lentiviral expression vector of HGFR-scFv was constructed successfully, which would help to study the important role of HGFR in following experiments.
authors.affiliation: Department of Clinical Laboratory, Suzhou Municipal Hospital, Suzhou 215000, China.
authors.forename: Yonghua
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Center for Bio-Pharmacy, Suzhou Institute, Tongji University, Suzhou 215000, China.
authors.forename: Jia
authors.initials: J
authors.lastname: Guo
authors.affiliation: Center for Bio-Pharmacy, Suzhou Institute, Tongji University, Suzhou 215000, China.
authors.forename: Yanxin
authors.initials: Y
authors.lastname: Yin
authors.affiliation: Center for Bio-Pharmacy, Suzhou Institute, Tongji University, Suzhou 215000, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Jiang
authors.affiliation: Department of Clinical Laboratory, Suzhou Municipal Hospital, Suzhou 215000, China.
authors.forename: Hongsheng
authors.initials: H
authors.lastname: Zhu
authors.affiliation: Department of Clinical Laboratory, Suzhou Municipal Hospital, Suzhou 215000, China.
authors.forename: Guodong
authors.initials: G
authors.lastname: Zhang
authors.affiliation: Center for Bio-Pharmacy, Suzhou Institute, Tongji University, Suzhou 215000, China.
authors.forename: Bingyu
authors.initials: B
authors.lastname: Li
mesh_terms: D000818:Animals; D000915:Antibody Affinity / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D003001:Cloning, Molecular / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006825:Hybridomas; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D008856:Microscopy, Fluorescence; D011485:Protein Binding / Q000276:immunology; D019859:Proto-Oncogene Proteins c-met / Q000276:immunology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D057127:Single-Chain Antibodies; D049452:Green Fluorescent Proteins; C491743:MET protein, human; D019859:Proto-Oncogene Proteins c-met