【摘要】 目的构建弓形虫ROP31基因重组真核表达载体,对其编码的蛋白进行生物信息学分析。方法提取弓形虫RH株速殖子总RNA,采用PCR扩增ROP31基因,构建其真核表达载体pET30a-ROP31。通过生物信息学软件结合在线程序分析ROP31基因编码蛋白的理化性质、抗原表位、空间结构等。结果 ROP31基因PCR扩增产物大小约1500bp,与预期一致,构建的重组质粒经双酶切鉴定表明目的基因插入正确。生物信息学分析ROP31基因编码蛋白有多个磷酸化修饰位点,具备多个跨膜区域;二级结构和空间结构分析ROP31蛋白与ROP家族优秀DNA疫苗ROP5、ROP17、ROP18在结构上相似;IEDB在线程序及DNA MAN软件分析ROP31蛋白具备比SAG1更为优秀的线性T、B细胞表位。结论成功构建重组真核表达载体pROP31,生物信息学分析其编码的ROP31蛋白具备优秀的细胞表位,这将为弓形虫ROP31DNA疫苗的研究提供理论依据。更多还原

【Abstract】 Objectives To construct a eukaryotic expression vector for the ROP31 gene in Toxoplasma gondii and to bioinformatically analyze the protein coded for by the ROP31 gene. Methods Total RNA was extracted from the tachyzoites of T.gondii,and the ROP31 gene was amplified using PCR.The recombinant eukaryotic expression plasmid pET30 a-ROP31 was constructed.The physical and chemical characteristics,epitopes,and spatial structure of the ROP31 protein were analyzed using bioinformatic software and online programs. Results Amplification of ROP31 with PCR yielded a product of about 1 500 bp,which was the expected size.The results of bioinformatic analysis indicated there are several phosphorylation sites and a transmembrane region in ROP31.The results of structural analysis indicated the structure of ROP31 was similar to that of ROP5,ROP17,and ROP18,which are used in several exceptional DNA vaccines.Results from the online program IEDB and the software DNAMAN indicated that ROP31 had a higher antigenic index than SAG1. Conclusion The eukaryotic expression vector pROP31 was successfully constructed.Results of bioinformatic analysis indicated that ROP31 had excellent epitopes.This could provide a theoretical basis for study of DNA vaccines using T.gondii ROP31.更多还原