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[Construction and verification of anti-MM scFv-tP fusion protein expression vector]
Hao Wang1, Yi-Fei Yang, Wei Wang
1Department of Dermatology, Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, China.E-mail: wanghaonxyc@163.com.
Objective:
To construct an expression vector of anti-MM scFv-tP fusion protein and test its expression efficiency and function.
Methods:
The truncated protamine (tP) gene sequence was added to the gene of single chain antibody against the specific antigen on the surface of malignant melanoma tumor cells using PCR. A GST-fusion expression vector was constructed and the soluable protein was expressed in the E.coli system. After cleavage and purification, the purified fusion protein was obtained. The binding activity of Anti-MM scFv-tP and siRNA was detected by EMSA. Flow cytometry and confocal microscopy were used to detect the cell surface antigen binding activity of the fusion protein.
Results:
The expression vector of Anti-MM scFv-tP fusion protein was successfully constructed. The soluable protein could be expressed in the E.coli system, and the purified fusion protein was obtained. The anti-MM scFv-tP fusion protein retained siRNA binding ability and could directly target malignant melanoma (MM) LiBr cells.
Conclusion:
The recombinant GST- Anti-MM-scFv-tp expression vector was successfully constructed. The fusion protein retains siRNA binding ability and can directly target LiBr cells to provide a reliable tool for further study.
