Abstract:

【Objective】 The present study aimed at preparation of monoclonal antibody against the recombinant PthA-NLS and isolaton of the relative ScFv (Single Chain Variable Fragment) genes, providing a possibility to better understand the pathogenesis mechanism via PthA, and to develop proper construct for future experimentation to obtain citrus plants resistant to citrus canker disease by transformation and plant antibody techniques. 【Method】 The recombinant polypeptide PthA-NLS was injected into Balb/c mice to produce monoclonal antibody. Total RNA was isolated from the hybridoma cell line 3D10H2 which secreted anti-PthA-NLS McAb with Trizol reagent, and the variable region genes were amplified with specific primers by RT-PCR and SOE-PCR (splicing by overlap extension), and then the ScFv gene was constructed. The recombinant ScFv gene was cloned into pGEM-T and pET32a(+) vector. The later plasmid was transferred into E. coli BL21(DE3) and the expression of the recombinant protein was induced. 【Result】 Three cell lines producing monoclonal antibody against PthA-NLS were acquired and named 1C8H1, 2D12B6 and 3D8A10. The recombinant ScFv gene of about 750 bp was constructed. The sequencing results showed that the ScFv gene consists of a 360 bp heavy chain, a 342 bp light chain and a 45 bp linker region. The recombinant fusion ScFv protein was expressed by IPTG induction, and a 44.5 KDa of recombinant fusion protein was obtained. 【Conclusion】 Three cell lines stably producing monoclonal antibody specifically bound to PthA-NLS were obtained, and the relative ScFv gene was constructed and successfully expressed in E.coli. These results may play an important role in further understanding of the pathogenesis mechanism and in the development of possible citrus resistant to canker disease by genetic transformation and plant antibiobody.

Key words: citrus canker, monoclonal antibody, ScFv antibody, pthA