Construction of the lentiviral expression vector for anti-p185(erbB2) mouse/human chimeric antibody.
- Author:
Fang LIU
1
;
Li LI
;
Wei ZHANG
;
Qi WANG
Author Information
1. Affliated Tumor Hospital of Guangxi Medical University, Nanning 530021, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Antibodies, Monoclonal;
biosynthesis;
genetics;
Cell Line;
Chimera;
Cloning, Molecular;
Humans;
Lentivirus;
genetics;
metabolism;
Mice;
Receptor, ErbB-2;
biosynthesis;
genetics;
immunology;
Recombinant Fusion Proteins;
biosynthesis;
genetics;
immunology;
Transfection
- From:
Journal of Biomedical Engineering
2013;30(2):347-354
- CountryChina
- Language:Chinese
-
Abstract:
This research was to construct the lentiviral expression vector for anti- p185(erbB2) mouse/human chimeric antibody and to determine the expression of the chimeric antibody gene in 293T cells transfected with this vector. The genes (vL and vH) coding light and heavy chain of variable regions of anti-p185(erbB2) mAb and the constant regions of human IgG1 (kappa and gamma1) were cloned with PCR method. The target genes were assembled by three-primers PCR method to obtain the chimeric light chain (L) and the chimeric heavy chain (H). Both chains inserted into the down stream and upper stream of IRES gene of the plasmid pVAX1/IRES respectively. We digested the plasmid pVAX1/ H-IRES-L with endoenzyme and subcloned H-IRES-L into the lentiviral vector pWPI. The enzyme digestion and sequence analysis showed that the lentiviral expression vector pWPI/H-IRES-L was constructed correctly. Then, it was transfected into 293T cells and after 48h, GFP protein expression in 293T cells were detected by fluorescent microscope and the chimeric antibody expression was detected by RT-PCR and direct ELISA. The results showed that after 293T cells were transfected with recombination plasmid, both light and heavy chains of the chimeric antibody genes could express together. The chimeric antibody expressed could bind to p185(erbB2) specifically. This research may lay a sound foundation for further study of anti-p185(erbB2) engineered antibody.
