摘要
目的构建甲型H1N1流感病毒HA基因的原核表达质粒,获得融合表达蛋白,并对表达蛋白的免疫反应性进行分析。方法人工合成A/California/05/2009 H1N1流感病毒的HA基因,以合成基因为模板,通过PCR方法扩增出去除信号肽的HA部分基因片段,然后将去除信号肽的HA基因克隆至pET30a(+)原核表达载体中,构建出原核表达质粒,再将重组质粒转化E.coliBL21(DE3)表达菌株;重组菌经IPTG诱导后,收集菌体进行SDS-PAGE电泳分析,Western blot分析表达产物的免疫反应性。结果获得了HA基因的原核表达重组菌,细菌经IPTG诱导表达后,SDS-PAGE分析可见约67.4 ku大小的目的蛋白表达条带,Western blot结果显示,表达产物与人甲型H1N1流感患者阳性血清具有反应性。结论成功表达出甲型H1N1流感病毒的HA蛋白,该蛋白具有良好的免疫反应性,为甲型H1N1流感的快速诊断方法的建立提供了生物材料。
In order to obtain fusion hemagglutinin （HA） protein of A/California/05/2009 H1N1, the recombinant prokaryotic expression vector for HA gene was constructed and the immunogenicity of its product was initially investigated in the present study. A pair of primers was designed according to the gene sequence of HA gene from A/California/05/2009 H1N1 in GenBank and amplified by PCR using synthetic DNA of HA as gene template. The purified HA was cloned into pET30a（＋） to constructed a prokaryotic expression plasmid, and the recombinant plasmid was transformed into competent bacteria of E. coli BL21 （DE3）. Then, the bacteria were induced by IPTG and their lysates were analyzed by SDS-PAGE and Western blot respectively. It was found that a 67.4 ku protein band was expressed by SDS-PAGE analysis. As demonstrated by Western blot, this expressed product showed the capacity of reacting with antibodies in human positive sera against influenza A virus subtype H1N1. All the results suggested that this expressed HA of H1N1 influenza virus would be very helpful in the development of rapid diagnosis methods.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2010年第10期923-925,948,共4页
Chinese Journal of Zoonoses
基金
国家自然科学自然基金项目(No.30871860)
江苏省自然科学基金项目(BK2008011)
江苏省青蓝工程项目联合资助
