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在成功克隆流感病毒H1N1全长HA(Hemagglulinin,HA)、NA(Neuramidinase,NA)基因并测序的基础上,将部分基因序列克隆到表达载体pMETA上,构建了重组表达质粒pMETA/HA(52~1557bp)、pMETA/NA(121~1263bp),电转化真核酵母菌pMAD16,甲醇诱导表达,利用Ni2+亲和层析柱对重组蛋白进行纯化,并用Western blotting和ELISA方法检测其抗原性。SDS-PAGE显示重组蛋白在酵母菌中可以高效表达,蛋白纯度占总蛋白的95%以上,ELISA和Western blotting实验证实,重组蛋白具有良好的抗原性。成功克隆和表达了流感病毒H1N1HA、NA基因序列,为流感病毒H1N1诊断试剂和疫苗的开发等进一步的研究提供了参考。
Based on the successful cloning and sequencing of the full-length HA (Hemagglulinin) and NA (NA) genes of influenza virus H1N1, the partial gene sequence was cloned into the expression vector pMETA to construct the recombinant expression plasmid pMETA / HA (52 ~ 1557bp) and pMETA / NA (121 ~ 1263bp) respectively. The eukaryotic yeast pMAD16 was transformed into E.coli. The recombinant protein was purified by Ni2 + affinity chromatography. The antigenicity was detected by Western blotting and ELISA. SDS-PAGE showed that the recombinant protein was highly expressed in yeast. The purity of the protein was above 95% of the total protein. ELISA and Western blotting confirmed that the recombinant protein had good antigenicity. The successful cloning and expression of the H1N1 HA and NA gene sequences of influenza virus provide a reference for further research on the development of H1N1 influenza vaccine and vaccine.