The Cloning and Prokaryotic Expression of Candidatus Liberibacter Asiaticus CLIBASIA_03230 Gene

Purpose Pathogenic bacteria secreted proteins play an important role in the process of pathogen infection. Analysis of the biological functions of secretory proteins will help to clarify the molecular mechanism of pathogen-plant interaction. In this study, the entire genome of Candidatus Liberibacter asiaticus was subjected to bioinformatics analysis, and CLIBASIA_03230 gene (named as Clsp11) was predicted to encode a secreted protein. Therefore, the Clsp11 was cloned and inserted into the prokaryotic expression vector, and the Clsp11 protein was expressed and purified for preparation of the specific antibodies in the next step.

Method Total DNA was extracted from Ca. L. asiaticus-infected periwinkle, and was used as template to amplify Clsp11. The resulting PCR products were cloned into pMD18-T vector and sequenced. With the In-Fusion cloning technique, the Clsp11 was inserted into the pET30a, resulting in pET30a-Clsp11. The pET30a-Clsp11 was subsequently transformed into Escherichia coli BL21 and induced by IPTG to express the recombinant protein Clsp11, the N-terminus of which was fused with 6×His tag. After analysis with SDS-PAGE, the Clsp11 recombinant protein was purified by nickel column.

Result The Clsp11 protein was successfully expressed, and reached high expression level when IPTG induction for 5 hours. Additionally, a large portion of the recombinant proteins present as inclusion bodies.

Conclusion In this study, the Clsp11 of Ca. L. asiaticus was cloned, the prokaryotic expression vector pET30a-Clsp11 was constructed, and the recombinant protein Clsp11 was expressed and purified, thus laying the foundation to prepare specific antibodies as well as investigate the biological function(s) of Clsp11.