【摘要】 根据GenBank公布的禽流感病毒H5N1亚型血凝素基因(HA)(GenBank:DQ023145)序列设计一对引物P1、P2,以重组质粒pUC-HA为模板扩增去除信号肽的HA成熟蛋白。PCR产物克隆入pMD18-T载体,经测序发现在967位A突变为T,形成一个终止密码子TAA。在突变位点附近设计两条有21个碱基配对的突变引物P3、P4,采用重叠延伸剪切法(SOE)用A定点替换T碱基,然后将正确的基因片段定向插入到表达载体pET-32a(+)中,诱导表达获得正确的表达产物。Western-blot分析表明,表达的重组蛋白能与经BL21(DE3)大肠杆菌菌体裂解液处理的H5亚型禽流感病毒阳性抗血清发生特异性反应。利用纯化的重组HA蛋白初步建立了检测H5亚型禽流感病毒抗体的间接ELISA方法,该方法可以代替传统的血凝与血凝抑制方法用于区分禽流感病毒的血清亚型。本研究为禽流感病毒亚单位疫苗及新型诊断试剂盒的研究奠定了基础。更多还原

【Abstract】 Using a pair of specific primers designed according to the relevant nucleotide sequence from GenBank (Accession number:DQ023145), the HA gene of AIV H5N1 subtype with the signal peptide sequence was obtained by PCR from the recombinant plasmid pUC-HA, which contains the full length of HA gene. Sequence analysis showed that a single base, an A, was replaced by aT to generate a stop codon (TAA) at position 967 of HA gene. Splicing by overlap extension ( SOE) method was applied to correct this mutation with two overlapped mutation primers P3 and P4, and then the correct HA gene was inserted into the expression vector pET-32a (+) to geerate the recombinant plasmid pET-HA-2. The target gene was successfully expressed in the host cell E.coli BL21(DE3) in inclusion bodies when induced with 1.0 mmol/L IPTG. Western-blot analysis proved that the recombinant protein had good immunoreactivity to positive serum of H5 subtype AIV. With this recombinant HA protein, an indirect ELISA method (iHA-ELISA) to detect antibodies of H5 subtype AIV was established. Interestingly, this iHA-ELISA could distinguish H5 positive serum from that of H7 and H9 subtypes. This results provide baseline for the investigating new vaccine and the development of new method to detect AIV.更多还原