MOLECULAR-CLONING AND EXPRESSION OF SALMONELLA-PARATYPHI A-52 KDA SPECIFIC PROTEIN GENE

Monoclonal antibodies (MAbs) specific to Salmonella paratyphi A have b een established by our group in 1989. These MAbs were proven to be spe cies-specific for 52 kDa protein of S. paratyphi A but the nature of t his protein is unknown. However, our group have proved that the 52 kDa protein which is specific to S. typhi was flagellin. This present stu dy has characterized the 52 kDa protein of S. paratyphi A and identifi ed its encoded gene. The plasmid containing the specific 52 kDa antige n gene was cloned from the S. paratyphi A genome, herein designated pS KA-4. Partial nucleotide sequences from this clone was analysed by com puter program and found to be phase 1-a flagellin gene of S. paratyphi A. In addition, the nucleotide sequence analysis from such clone also showed that the structural gene for phase 1 flagellin has amino acid sequences conserved at the terminal whereas the central region is vari able among Salmonella spp. Therefore, the central portion of flagellin which highly polymorphic in amino acid sequences would be the most sp ecific to S. paratyphi A, mino acid sequences would be the most specif ic to thus, should be used as specific antigen for developing specific diagnosis of S. paratyphi A infection. Using the PCR technique, an ex pression plasmid containing the antigen gene producing only the variab le region in the central portion of flagellin from S. paratyphi A, nam ely pSKA-7, has been established. The recombinant protein produced by the established plasmid has a MW 33.5 kDa as detected by immunoblottin g using specific MAbs. Further study by using this specific flagellin protein for immunodiagnosis of S. parathyphi A infection is being carr ied out in our laboratory.