Protokaryotic Expression and Polyclonal Antibody Preparation of OSVDAC5 Protein in Rice YTB

Objective: To clone osvdac5 gene from rice YTB and expression in E.coli,and prepare the polyclonal antibody with the purified OSVDAC5 protein.Method:The total RNA was extracted from YTB seedlings with Trizol,and the cDNA was reverse transcripte.The osvdac5 gene ORF amplified through PCR was linked with pET-30a to construct the recombinant plasmid pET-30a-osvdac5.The OSVDAC5 protein was expressed induced with IPTG in E.coli strain BL21,and checked by SDS-PAGE.The purified OSVDAC5 protein through Nickel column was used to prepare the polyclonal antibody.The specificity of the polyclonal antibody for OSVDAC5 was checked by Western Blot.Result:The length of osvdac5 gene ORF cloned into the prokaryotic expression vector was 813 bp,which containing 271aa.In E.coli strain BL21 the optimal condition for OSVDAC5 protein expression was under 0.7mmol/L IPTG at 15℃ with 17 hours.The OSVDAC5 fusion protein existed as inelusion body,insoluble form.The purified OSVDAC5 protein was a single band with about 30 kD.The results from Western Blot analysis showed that the polyclonal antibody from OSVDAC5 combined specificity with OSVDAC5.Conclusion: Succession of cloning and expression of the osvdac5 gene from YTB.The polyclonal antibody from OSVDAC5 showed definite specificity with OSVDAC5 protein.These laid a foundation for studying the expression mode of OSVDAC5 during different growth and development stage in plant.