Objective　To further understand the mechanism of PBP2a mediated the drug-resistance of methicillin-resistant Staphylococcus aureus(MRSA) with a bacterial two hybrid system. 　Methods　The fragment encoded PBP2a TPase domain of MRSA strain N315 was amplified by PCR and then cloned into pRBR vector to construct the bait plasmid pBR-PBP2a. The genomic DNA of MRSA strain N315 was partially digested with Sau3AⅠ and the fragments between 500 to 1 500 bp were recovered

then ligated to pRAC vector prior cut with BamHⅠ to get a genomic DNA library. The bacterial two hybrid system was constructed by transforming the plasmids of the DNA library into KS1 host cells (harboring a LacZ reporter gene) prior transformed with the bait plasmid pBR-PBP2a and the prey plasmids were screened out by LacZ test. After isolation and DNA sequencing of the prey plasmids

the inserts were characterized by bioinformatic analysis and the peptide that interacts with PBP2a were obtained. 　Results　The genomic DNA library of MRSA strain N315 and the bait plasmid pBR-PBP2a were successfully constructed. Nine clones were obtained with more than 2 times raised for LacZ activity in the bacterial two hybrid assay and their insert sequences were qualified by DNA sequencing. The longest insert was 648 bp and the shortest one was 334 bp. There were 14 genes related with the 9 inserts and 10 of them encoded proteins with unknown functions. Among the 4 familiar genes

one encoded dihydrodipicolinate synthase

one encoded dihydrodipicolinate reductase and the others encoded chromosome segregation SMC proteins. The peptides that may interact with PBP2a in the 9 prey plasmids composed of 14 to 46 amino acids.  　Conclusion　The proteins interact with PBP2a are successfully screened by the bacterial two-hybrid system assay

which facilitates further understanding the mechanism of PBP2a mediated the drug-resistance in MRSA.