Based on prokaryotic expression vector pCold TF, a new vector pL118 was constructed by replacing the His-Tag sequence of the original vector with the restriction enzyme site of Spe I in molecular biological methods. His-Tag sequence was introduced to the 3′ end of target gene through PCR primer, and this method facilitated the expression of fusion protein and the removal of solubilization tag from the target protein. The fusion protein human CD81-Trigger Factor (TF) was expressed and purified. Human CD81 protein was obtained by removing the TF solubilization tag by proteinase Factor Xa. The purified human CD81 protein laid the foundation for the selection of targeted drugs, making antibody and exploring the function of CD81. The construction of the new expression vector pL118 and the expression and purification of human CD81 protein have provided a new method for obtaining insoluble protein based on the prokaryotic expression system.