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Archive > Volume 22 Issue 5 > 2015,22(5):603-606. DOI:10.3872/j.issn.1007-385X.2015.05.009 Prev Next
Basic Research
Construction and expression of a third generation chimeric antigen receptor target epidermal growth factor receptor variant Ⅲ-specific
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Abstract:
Objective:To design and construct a vector expressing a human epidermal growth factor receptor variant Ⅲ (EGFRvⅢ)-specific third generation chimeric antigen receptor (CAR) for potential use in adoptive immunotherapy of cancer. Methods:An anti-EGFRvⅢ CAR, referred to as EGFRvⅢ/3CAR, comprising the variable fragment ( EGFRvⅢscFv), hinge region/transmembrane region, CD28 & CD137 intracellular signal region, and CD3ζ chain of a monoclonal antibody against human EGFvⅢ was generated and inserted in frame into pCDH-CMV-MCS-EF-copGFP EcoR1 and BamH1 sites. The integrity of the resultant recombinant vector was confirmed by restriction enzyme mapping and sequence analysis. For further functional integrity verification, the recombinant vector was transfected into 293T cells and the fusion protein expression was assessed by Western blotting. Results:DNA sequencing and restriction enzyme mapping demonstrated the sequence of EGFRvⅢ/3CAR was corret. Western blotting detected EGFRvⅢ/3CAR protein at approximately 58 kD in 293T cells transfected with the EGFRvⅢ/3CAR construct. Conclusion: We have successfully designed and constructed a recombinant vector expressing a human EGFRvⅢ-specific third generation CAR, which may prove usefull in further studies on CAR-based cancer immunotherapy.
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Project Supported:
Project supported by the National Natural Science Foundation of China（No. 81172415），and the Scientific Foundation of National Health and Family Planning Commission of China (No.201301010)
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