Zhang Ye, Yu Zaijiang, Xin Li, Chen Yongkun, Tang Qihui, Chen Yubao, Chen Qingxuan, Shu Yuelong
Department of Influenza, Nattonat Institute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China.
Sheng Wu Gong Cheng Xue Bao. 2010 Aug;26(8):1068-73.
On the basis of successful cloning the full length hemagglulinin (HA) and neuramidinase (NA) gene and sequence analysis of influenza virus H1N1, part of the gene was ligated into pMETA. Expression vectors pMETA/HA (52-1 557 bp) and pMETA/NA (121-1 263 bp) were constructed and expressed in pMAD16 induced by methanol. Recombinant protein was purified through Ni2+ affinity chromatography. Western blotting and ELISA were used to determine the antigenic activity of the recombinant protein. SDS-PAGE showed that the recombinant capsid gene could be overexpressed in Pichia methanolica. ELISA and Western blotting showed that the recombinant protein had antigenicity.
在成功克隆甲型H1N1流感病毒全长血凝素（HA）和神经氨酸酶（NA）基因并进行序列分析的基础上，将部分基因连接到pMETA中。构建了表达载体pMETA/HA（52 - 1557 bp）和pMETA/NA（121 - 1263 bp），并在甲醇诱导的pMAD16中表达。重组蛋白通过Ni2+亲和层析进行纯化。采用蛋白质免疫印迹法（Western blotting）和酶联免疫吸附测定（ELISA）来测定重组蛋白的抗原活性。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）显示重组衣壳基因可在甲醇毕赤酵母中过表达。ELISA和Western blotting表明重组蛋白具有抗原性。
