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JZTX-V, an inhibitor of voltage-gated sodium and potassium channels, is derived from the venom of the spider Chilobrachys jingzhao in China. JZTX-V was synthesized using a solid-phase chemical approach with Fmoc-protected amino acids to explore its function further. The synthetic peptides were purified using reverse-phase high-performance liquid chromatography (RP-HPLC) and then subjected to oxidative refolding under optimal conditions. A unique peak was observed in the RP-HPLC chromatogram for refolded JZTX-V, and the ratio to native JZTX-V was 1:1 for the mixed samples. Subsequently, the analgesic potential of the synthetic peptide was evaluated in mouse models of pain. In the Formarin model, JZTX-V significantly reduced pain scores in 60 min and its efficacy was comparable to that of morphine. JZTX-V also exhibited excellent analgesic effects in models of postoperative pain and mechanical allodynia. However, JZTX-V had no effect on thermal stimulation injury in the hot plate experiment and did not affect motor coordination. These results indicate that JZTX-V effectively alleviates inflammatory pain in animals and provides a promising template for the design of future clinical analgesic drugs. [ABSTRACT FROM AUTHOR]