三七病程相关蛋白1基因的克隆与表达分析  

Cloning and expression analysis of pathogenesis-related protein 1 gene of Panax notoginseng

作　　者：李瑞博[1,2] 崔秀明[2] 刘玉忠[1] 吴志刚[1] 林淑芳[1] 申业[1] 黄璐琦[1] 

机构地区：[1]中国中医科学院中药资源中心,北京100070 [2]昆明理工大学生命科学与技术学院,云南昆明650000

出　　处：《药学学报》2014年第1期124-130,共7页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81130070;81173490)

摘　　要：在生物信息学分析基础上,采用逆转录聚合酶链式反应(RT-PCR)方法从三七中获得病程相关蛋白1(pathogensis-related protein 1,PR1)基因的开放阅读框,命名为PnPR1,测序结果显示该序列长501 bp,编码166个氨基酸,其蛋白质分子质量为18.1 kD。利用NCBI/Blastp和BioEdit软件进行同源性比对显示,PnPR1基因编码蛋白与葡萄、烟草、番茄等高等植物中的PR1蛋白同源性较高,且具有相同的富含半胱氨酸蛋白的保守结构域。将构建的重组载体pET28a(+)-PnPR1在宿主菌Escherichia coli BL21中经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达融合蛋白,在不同诱导时间、诱导温度、IPTG诱导浓度和IPTG添加时间下对诱导条件进行优化。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析结果表明,PnPR1基因编码蛋白的最佳诱导条件为:IPTG终浓度0.4 mmol·L-1、IPTG添加时间为转接后4 h、诱导温度28℃、诱导时间20 h。这为蛋白纯化及单克隆抗体的制备奠定了一定的基础。By reverse transcription-polymerase chain reaction （RT-PCR）, an open reading frame of pathogenesis-related protein 1 （PR1） was isolated from Panax notoginseng and named as PnPR1. Molecular and bioinformatic analyses of PnPR1 revealed that an open reading frame of 501 bp was predicted to encode a 166-amino acid protein with a deduced molecular mass of 18.1 kD. Homology analysis showed that the deduced amino acid sequence of PR1 protein of Panax notoginseng had a high similarity with other higher plants had the same conservative structure domain of cysteine-rich secretory protein （CAP）. The recombinant expressed plasmid pET28a（＋）-PnPR1 was expressed in Escherichia coli BL21. The expression conditions were optimized by induction at different times, different temperatures, different IPTG concentrations and different giving times. The optimum expression condition was 0.4 mmol.L-1 IPTG at 28 ℃ for 20 h. The successful expression of PnPR1 provides some basis for protein purification and preparation of the monoclonal antibody.

关 键 词：三七 病程相关蛋白 生物信息学分析 原核表达 条件优化 

分 类 号：R931[医药卫生—生药学]