Clone, expression and identification of penicillin binding protein 2a of methicillin-resistant Staphylococcus aureus isolated from patients.
- Author:
Yan DONG
1
;
Guo-Fu DING
;
Bin LI
;
Sheng-Qi HE
;
Wei YAN
;
Hong ZHOU
;
Xian-Yuan WANG
Author Information
- Publication Type:Journal Article
- MeSH: Base Sequence; Cloning, Molecular; Gene Expression; Humans; Methicillin Resistance; genetics; Methicillin-Resistant Staphylococcus aureus; genetics; isolation & purification; Microbial Sensitivity Tests; Penicillin-Binding Proteins; genetics; metabolism; Peptide Synthases; genetics; metabolism; Plasmids
- From: Chinese Journal of Burns 2007;23(2):100-103
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo 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.
METHODSAccording 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.
RESULTSThe 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.
CONCLUSIONThe soluble PBP2a of MRSA isolated from patients is expressed successfully by gene recombinant technology.
