[Clone, expression and identification of penicillin binding protein 2a of methicillin-resistant Staphylococcus aureus isolated from patients].
OBJECTIVE
To clone, express and identify the mecA fragment which encoded penicillin binding protein 2a (PBP2a) from methicillin-resistant staphylococcus aureus (MRSA) isolated from patients by gene recombination method.


METHODS
According to the sequence of mecA gene recorded in GenBank, the primer of mecA fragment which encoded amino acids 25 - 668 of PBP2a was designed. Then the mecA fragment was amplified by PCR and cloned into pQE30 plasmid. After being identified by enzyme digestion and sequencing, the recombinant plasmid was transferred into E. coli M15 [pREP4], and then its expression was induced by 1 mmol/L Isopropy-beta-D-Thiogalactoside (IPTG). The expression product was analyzed by SDS-PAGE, protein sequencing and mass spectroscopy.


RESULTS
The recombinant pQE30- mecA had been successfully constructed. The result of sequencing showed that the mecA fragment had 1932 bases, including 9 bases undergoing mutation. After being induced for 6 hours by IPTG, the soluble protein in M15 (pQE30- mecA), with a relative molecular weight of 74 x 10(3), was found by SDS-PAGE. The soluble protein had been confirmed to be PBP2a after identification.


CONCLUSIONS
The soluble PBP2a of MRSA isolated from patients is expressed successfully by gene recombinant technology.
Dong
Yan
Y
Department of Pharmacology , the Third Military Medical University , Chongqing 400038, P. R. China.
Ding
Guo-Fu
GF
Li
Bin
B
He
Sheng-Qi
SQ
Yan
Wei
W
Zhou
Hong
H
Wang
Xian-Yuan
XY
D001483:Base Sequence; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D016106:Methicillin Resistance / Q000235:genetics; D055624:Methicillin-Resistant Staphylococcus aureus / Q000235:genetics* / Q000302:isolation & purification; D008826:Microbial Sensitivity Tests; D046915:Penicillin-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids
D046915:Penicillin-Binding Proteins; C478699:penicillin-binding protein 2a, Streptococcus; D010453:Peptide Synthases