As an important member of the mitogen-activated protein kinase (MAPK) family, MEKK3 is mainly involved in the activation of c-jun N-terminal kinase(JNK) and extracellular regulated protein kinases (ERK).In this study, bioinformatics methods were used to analyze the physical and chemical properties, hydrophilicity/hydrophobicity, transmembrane region, secondary structure, tertiary structure and interaction protein of wild-type MEKK3 (MEKK3WT) and inactivatedMEKK3(MEKK3K391M).The results showed that the stability of MEKK3K391M protein was enhanced, the strongest hydrophilic and hydrophobic sites were not changed, the α-helix region, β-folding and protein binding sites were different from MEKK3WT, and the tertiary structure analysis showed that the steric hindrance of MEKK3K391M increased.According to the cDNA sequence of human MEKK3 (NM_203351) provided by GenBank, the target gene of wild-type MEKK3 (MEKK3WT) was amplified, and the target gene of the kinase inactivated MEKK3 (MEKK3K391M) was amplified by overlapping extended PCR site-directed mutation technology, and cloned into the eukaryotic expression vector pcmv-tag-2c with FLAG tag. MEKK3K391M gene sequence analysis results showed that the lysine (K) was mutated into methionine (M) at site 391.Western blot analysis showed that MEKK3WT and MEKK3K391M were highly expressed in PC12 cells. Biological activity test results showed that MEKK3WT could activate JNK and cause phosphorylation reaction of JNK, and its band gray value was significantly higher than that of the control group and the MEKK3K391M group.The P-JNK of MEKK3K391M group was not significantly different from that of the control group. In conclusion, the biological characteristics of MEKK3WT and MEKK3K391M were analyzed by bioinformatics software in this study, and the eukaryotic expression vectors of MEKK3WT and MEKK3K391M were constructed. providing useful tools for further functional studies of MEKK3 in the neurological diseases.