Abstract: The expression vector was constructed by plasmids containing SUMO,IF2,GST,NusA,MsyB,Trx and MBP fusion tags,which was transformed into E.coli Transetta(DE3) strain,to improve the expression yield of T4 DNA ligase(T4 DL).Then they were induced expression by auto-induction. The soluble expression of the fusion protein was efficiently and accurately detected by magnetic beads.The results showed that the yield of soluble fusion protein Trx-T4 DL induced expression from recombinant bacteria Transetta(DE3) (pNBE VⅡ-T4 DL) was the highest.Its solubility was greatly improved after the culture conditions were optimized,and the optimal induction conditions were 30℃,bottling volume 50 mL/250 mL,inoculation amount 2%,pH7. Meanwhile, the soluble T4 DL in the supernatant after disintegrating recombinant bacteria was purified by Ni⁺ column and MagNi magnetic beads,respectively.The high purity T4 DL was efficiently purified by MagNi magnetic beads,and its concentration was 1700.462 mg/L.Comparing with T4 DL activity of other companies,the enzymatic activity was determined as 500 U/mL, then it was successfully applied to the construction of low background cloning vector. It could provide a theoretical basis for the production and application of fusion protein Trx-T4 DL.