Abstract: Objective To construct recombinant vector pET-29a-CD63-LEL, induce protein expression, identify the expression product and purify the protein, and provide guidance for studying how CD63-LEL affect the invasion and metastasis of tongue squamous carcinoma. Methods The cDNA sequences of CD63-LEL in TCA-8113 cell were detected from NCBI and obtained by RT-PCR, identified by PCR, restriction enzyme digestion and DNA sequencing, and the recombinant plasmids were transformed into E.coli BL21 (DE3). Then, the pET-29a-CD63 -LEL fusion protein was induced by Isopropyl β-D-1-Thiogalactopyranoside (IPTG) and the pET-29a-CD63 -LEL fusion protein was obtained with the method of NI-NTA agarose. Results The recombinant vector pET-29a-CD63-LEL was constructed successfully. SDS-PAGE detection showed that adding 1mmol/L of IPTG in 37℃ with 12 h induction was the best condition to express pET-29a-CD63-LEL protein, the protein could be eluted under the condition of imidazole concentration (MCAC - 80/100/200/500).Western blot detected that the protein was located at 26 kU and 35 kU which was purified by Ni- NTA agarose. Conclusion pET-29a-CD63-LEL can be largely expressed in E.coli and relatively pure pET-29a-CD63-LEL protein can be achieved by Ni-NTA agarose purification.