禽流感病毒H5N1血凝素基因和神经氨酸酶基因在大肠杆菌中的表达

目的：克隆、表达和鉴定禽流感病毒H5N1血凝素基因（hemagglutinin，HA）和神经氨酸酶基因（neuramidinase，NA）序列,为制备抗体和基因工程疫苗打下基础。方法：在成功克隆禽流感病毒H5N1全长HA、NA基因并测序的基础上，将部分基因序列克隆到表达载体pET32a（＋）上，全基因序列克隆到表达载体pGEX4T-1上，构建了重组表达质粒pET32a（＋）/HA(49~1587bp)、pET32a（＋）/NA(121~1141bp)、pGEX4T-1/HA、pGEX4T-1/NA，转化大肠杆菌BL21/rosetta，IPTG诱导表达，利用Ni2＋亲和层析柱和GSTra P4B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。结果：重组蛋白在大肠杆菌中可以高效达,SDS PAGE显示其相对分子质量与预计大小一致，蛋白纯度占总蛋白的90％以上。ELISA和Western blotting实验证实，重组蛋白具有良好的抗原性。结论:成功克隆和表达了禽流感病毒H5N1 HA、NA基因序列,为禽流感病毒H5N1诊断试剂和疫苗的开发等进一步的研究奠定了基础。

Expression of the Hemagglutinin and Neuramidinase Gene of Influenza A Virus H5N1 in E. coli

Objective: To clone, express and characterize the HA (hemagglutinin) and NA (neuramidinase, NA) protein of avian influenza virus H5N1. Methods:On the basis of successful clone the full length HA and NA 0gene and sequence analysis of avian influenza virus H5N1 ,Ligated part of the gene into pET32a(+). full of the gene into pGEX4T-1. An expression vector pET32a(+)/HA(49 ～ 1587bp) ,pET32a(+)/NA(121 ～ 1141bp), pGEX4T-1/HA,pGEX4T-1/NA were constructed and expressed in E. coli BL21/rosetta induced by IPTG. recombinant protein was purified through Ni~(2+) and GSTrap 4B affinity chromatography column. Western blotting and ELISA were used to determine the antigenic of the recombinant protein. Results :The recombinant capsid gene can be overexpressed in E. coli. SDS-PAGE showed that the gene could express product as same as I might expect. The purity of the protein is greater than 90%. ELISA and Western blotting showed that the recombinant protein has good antigenic. Conclusion:The HA and NA protein of avian influenza virus HSN1 has been successful cloned and expressed, which could be useful for developing diagnose reagents or vaccine of H5N1.