甲型流感病毒核蛋白的表达与鉴定及其B细胞表位的预测

摘要

目的 构建甲型流感病毒核蛋白(nucleoprotein,NP)的原核表达载体,诱导NP融合蛋白在大肠杆菌中的表达与鉴定；预测人NP蛋白的B细胞抗原表位.方法 以pcDNA3.1(+)/NP为模板,利用PCR方法扩增目的片段NP,再将此片段插入原核表达载体pET-32a(+),并进行PCR鉴定、酶切鉴定和测序鉴定,将鉴定正确的质粒转入表达菌Rosetta-gami(2)中,用异丙基-D-硫代半乳糖苷(IPTG)诱导融合蛋白的表达,用SDS-PAGE和Western Blot检测表达产物.按Chou-Fasman和Gamier-Robson方法预测其编码蛋白的二级结构,采用Karplns-Schulz方法预测蛋白骨架区的柔韧性；用Kyte-Doolittle方法预测其亲水性,Emini方法预测蛋白质表面可能性及Jameson-Wolf方法预测抗原性指数.结果 成功构建了流感病毒核蛋白(NP)的原核表达载体,经SDS-PAGE检测NP蛋白得到了表达,Western Blot证明表达的重组蛋白具有免疫活性.在蛋白质第6～11、79～91、241～248和319～326区段附近很可能为B细胞表位优势区域.结论 流感病毒核蛋白的表达及B细胞表位的预测为流感病毒的快速诊断试剂的研发工作提供了基础.%Objective To construct the prokaryotic expression vector for the nucleoprotein (NP) of influenza A virus and express it in Escherichia coli,as well as to predict and analyze its B cell epitopes.Methods The complete NP gene was amplified from plasmids pcDNA3.1 (+)/NP by PCR and was inserted into prokaryotic expression plasmid pET-32a(+) to construct pET-32a (+) / NP.After identification by restriction enzymes digestion,PCR and sequencing analysis,pET-32a(+)/NP was transformed into E.coli Rosetta-gami(2) cells to express with induced by IPTG.Standard SDS-PAGE and Western blot method were applied for the fusion protein identification.The secondary structure and flexible regions of nucleoprotein were predicted by the methods of Chou-Fasman,Gamier-Robson and Karplus-Schulz.Moreover,hydrophilicity plot,surface probability plot and antigenic index of nucleoprotein were predicted by the methods of Kyte-Doolittle,Emini and Jameson-Wolf,respectively.Results Expression vector pET-32a(+)/NP was successfully constructed.It was showed by SDS-PAGE that The NP was expressed well in E.coli Rosetta-gami(2).Western Blot proved that the recombinant protein has immunological activity.The B cell epitopes in nucleoprotein possibly localized in the regions of 6-11,79-91,241-248 and 319-326.Conclusion The NP gene of influenza virus was successfully cloned and expressed.And the B cell epitopes were predicted.The study laid a solid foundation for development of a novel immunological diagnostic kit for influenza virus.