Transformation of HCMV pp150 gene into horsebean and it's identification
Objective To develop a human cytomegalovirus (HCMV) oral vaccine. Methods Gene fragment encoding the antigenic determinants of PP150 was amplified from HCMV AD169 by PCR using a pair of primers based on the previously published gene sequence for HCMV PP150. The Kozak sequence was placed up- stream of the start codon of pp 150 gene and KDEL sequence was added to the end of the gene. The modified gene was integrated into a vector pBI121. Then pp150 gene with 35S promoter and NOS terminator was excised from pBI121/pp150 to construct plant expression plasmid with pCAMBIA1300. The plant expression plasmid pCAM- BIA1300/pp150, which contains hygromycin photransferase gene as selection marker, was transferred into Agrobac- erium tumefaciens EHA105. The leaves of horsebean were infected with Agrobacerium tumefaciens, and calli were screened on medium containing hygromycin. Five hygromycin resistant plants were regenerated through somatic em- bryogenesis from embryogenic calli, 3 of which were identified as positive by PCR. Southern blot analysis showed that pp150 gene had been integrated into genomes of the 3 transgenic plants. The expression of pp150 gene in the 3 transgenic plants was further analyzed by RNA dot blot. Protein from pp150 transgenic horsebean was tested by ELISA and Western blot. Results PP150 proteins expressed in the 3 transgenic plants possess imrnunogenicity. Conclusion These transgenic plants will provide new material for the development of edible vaccines against HC- MV infection.