Abstract: AIM: To establish the eukaryotic expression vector of single-chain antibody fragments reacting with human CD₁₃ and scorpion toxin polypeptide analgesic antitumoral peptide (AGAP) and the target lethal effect to NB4 cells by genetic engineering method. METHODS: Buthus martensii Karsch active peptide AGAP gene was inserted eukaryotic expression vector pSecTag2 /CD₁₃ /RFP, and the recombinate eukaryotic expression vector pSecTag2 /CD₁₃ /AGAP was obtained. After double enzyme digestion and DNA sequencing identification, the vector was transfected into 293T cell line, the AGAP recombinant gene and protein expression were detected by RT-PCR and western blot methods. The recombinant protein was purified by ProBond(tm) Nickel-Chelating Resin kit and used to treat NB4 cells. NB4 cell cytoactive was measured by CCK-8 and the cell cycle was analyzed by FCM. RESULTS: The eukaryotic expression vector pSecTag2/ CD₁₃ /AGAP was successfully established by double enzyme digestion and DNA sequencing identification. AGAP gene and protein expression were detected by RT-PCR and Western Blot after the vectors were transfected into 293T cells. After treatment with the purified recombinant protein CD₁₃-AGAP, NB4 cells viability were decreased and CD₁₃-AGAP recombinant protein arrested NB4 cell cycle in G₁ phase and decreased in S phase. CONCLUSION: The recombinant plasmid pSecTag2 /CD₁₃ /AGAP was successfully established and expressed in 293T cells, the recombinant protein CD₁₃- AGAP had lethal effect to NB4 cells.

Key words: Buthus martensii Karsch, scorpion venom active peptides, analgesic antitumoral peptide, CD₁₃ single chain fragments, fusion protein