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

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。

Expression and identification of Nucleoprotein (NP) of Influenza A Virus and Prediction B cell epitopes at NP

Objective To construct the prokaryotic expression vector for the nucleoprotein （NP） of influenza A vi- rus 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. Stand- ard 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 meth- ods of Kyte-Doolittle, Emini and Jameson-Wolf, respectively. Results Expression vector pET-32a（＋）/NP was success- fully 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 local- ized 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 no- vel immunological diagnostic kit for influenza virus.