Abstract: Objective: To construct the lentivirus-based eukaryotic expression vector pLenti6/V5-TOPO-Tβ R IIDNglytk. Methods: Tβ R II DN was obtained from plasmid pAdTrack-CMV by PCR, and it was then connected withHSV-tk by recombinant PCR. After agarose gel electrophoresis was performed to validate the size of the product, the bandcontaining Tβ R II DNglytk was retrieved and was cloned into Lentiviral vector pLenti6/V5-TOPO using the TOPO cloningtechnique. Results: We have demonstrated successful cloning of Tβ R Ⅱ DNglytk into the Lentiviral vector pLenti6/V5-TOPO. The sequencing map of the plenti6/v5-D-TOPO-Tβ R Ⅱ DNglytk vector was consistent with theoretical predic-tions. Conclusion: Recombinant PCR combined with the TOPO cloning technique is a simple, highly efficient and rapidway to construct vectors. We used this method to produce a Lentivirus-based vector containing Tβ R Ⅱ DNglytk.