AIM: To construct and analyze eukaryotic expression plasmid inserted by Cx50 with V64G mutation through bioinformatics software. METHODS: The full coding domain sequence of Cx50 with V64G mutation was acquired from the blood of patients with cataract and was cloned into pcDNA3.1/Amp (+).The constructed plasmid was identified with PCR , enzyme digestion and sequencing. The analysis of Cx50 with V64G mutation was performed with bioinformatics software. RESULTS: Cx50 with V64G mutation was successfully amplified and its eukaryotic expression plasmid was constructed. Valine-64 is well conserved in the first extracellular loop of connexin 50 in different species and also in different human α-type gap junctional proteins. CONCLUSION: The successive reconstruction and verification of eukaryotic expression plasmid containing Cx50 with V64G mutation established the foundation for further studying the mechanism of cataract.