MRSA-PBP2a蛋白的体外诱导、抗体制备及鉴定

Induction of expression of MRSA-PBP2a protein in vitro and its antibody preparation

目的:探讨耐甲氧西林金黄色葡萄球菌(methicillin resistantstaphylococcusaureus,MRSA)蛋白的体外诱导;抗MRSA PBP2a(pencillinbindingprotein2a,PBP2a)蛋白抗体的制备及效价鉴定.方法:采用NCCLS推荐的琼脂板稀释法和PCR法筛选MRSA,以β内酰胺抗生素作为诱导剂,进行体外诱导产生PBP2a蛋白.免疫家兔后产生抗MRSA PBP2a蛋白抗体,采全血分离血清,用改良双向免疫扩散法进行抗体滴度测定.结果:11株临床分离的金葡菌采用二种方法均检测出2株MRSA,SDS PAGE和Westernblot结果均显示从MRSA菌株中均能诱导出了PBP2a蛋白;免疫后均能产生较高的效价抗体,分别为1∶32和1∶64.结论:成功诱导出产生耐甲氧西林金黄色葡萄球菌PBP2a蛋白菌株,并制备获得PBP2a蛋白,以及制备抗耐甲氧西林金黄色葡萄球菌PBP2a蛋白抗体,为研究MRSA PBP2a蛋白活性、MRSA的免疫治疗和MRSA快速鉴定方法学的建立奠定基础.

AIM: To induce the expression of MRSA-PBP2a protein in vitro and to prepare its antibody against MRSA-PBP2a. METHODS: Methicillin-resistant Staphylococcus aureus (MRSA) were isolated by agar’s slides according to National Committee for Clinical Laboratory Standards (NCCLS) protocols and polymerase chain reaction (PCR). The expression of MRSA-PBP2a was induced in vitro by adding-Lactam antibiotics. MRSA-PBP2a was purified and was used to immune rabbits and the antiserum was identified by double immunodiffusion. RESULTS: Two strains of MRSA were isolated using the agar’s slides and PCR. SDS-PAGE and Western blot analysis revealed the successful induction of PBP2a protein expression. Antibodies were detected in antiserum in 1∶32 and 1∶64 dilutions. CONCLUSION: MRSA-PBP2a is induced to express in vitro and is purified successfully. The antibodies against MRSA-PBP2a are prepared successfully, which lays the foundation for further study on MRSA-PBP2a activities, MRSA immunotherapy and MRSA identification.