金黄色葡萄球菌肠毒素A成熟肽基因序列的克隆及抗原性分析  

Cloning and antigenic analysis of the coding sequence of mature Staphylococcus aureus enterotoxin

作　　者：谢洋[1] 陶爱林[2] 欧阳顺林[1] 张建国[1] 伍秋容[2] 

机构地区：[1]510260,广州医学院第二附属医院耳鼻喉头颈外科 [2]510260,广州医学院第二附属医院变态反应科

出　　处：《中华生物医学工程杂志》2008年第1期-,共4页Chinese Journal of Biomedical Engineering

基　　金：国家自然科学基金，广东省科技厅科技计划

摘　　要：目的 扩增金黄色葡萄球菌 (金葡菌)肠毒素A(SEA)基因并对其进行抗原性分析,为后续抗原性弱化及安全性免疫毒素的构建提供依据.方法 以金葡菌基因组DNA为模板,以SEA编码基因特异区段为引物,采用Touchdown PCR克隆SEA成熟肽编码基因,经菌落PCR筛选出阳性克隆后再进行DNA测序及BL2SEQ比对分析,并应用生物信息学软件对所得基因编码蛋白各区段的抗原性进行分析.结果PCR产物克隆后,菌落PCR挑选得到阳性克隆,DNA测序后进行的BL2SEQ比对显示,所得序列与GenBank中登录的序列之间一致性达100%.SEA成熟肽各区段上的抗原性均值较高,同时,有几个突出高值点分布在不同的区段上.结论 应用特定的生物信息学方法 从理论上确定了SEA成熟肽的高抗原性位点,为后续弱化SEA的抗原性研究提供了依据.Objective To clone the coding sequence of Staphylococcus aureus enterotoxin A (SEA) and to analyze its antigenicity for attenuation in targeted immunotherapy of tumor.Methods Genomic DNA extracted from Staphylococcus aureus 26079 was used as template.The coding special region of SEA was used as primer. Touchdown PCR method was employed to clone the coding region of mature SEA. PCR product recovered was subcloned into the pGEM-T vector. Positive clones were validated and selected by colony PCR followed by further confirmation by DNA sequencing and BL2SEQ alignment. The antigenicity of the coded peptide was analyzed by bioinformatic software.Results Following PCR product recovery and cloning,several clones were positively confirmed by colony PCR. DNA sequence alignment showed that the clones were of 100% identity with that of SEA registered in GenBank. Online analysis by SYFPEITHI showed that relatively high antigenicity was harbored in the whole region of mature SEA peptide with some extremely high peak values scattered in different regions.Conclusions The gene fragment encoding SEA mature peptide is successfully cloned. It is necessary to confirm all the loci with high peak antigenicity in order to attenuate the antigenicity of the peptide.

关 键 词：葡萄球菌 金黄色 肠毒素类 基因克隆 抗原性变异 

分 类 号：R378[医药卫生—病原生物学]