摘要
根据已知H5N1亚型禽流感病毒血凝素(HA)基因序列设计、合成克隆引物.自灭活的云南地方H5N1亚型病毒阳性临床组织样品中提取总RNA,反转录后采用高可信度DNA聚合酶(PyobestTMDNA Poly-merase)扩增HA基因,采用Invitrogen定向表达系统(ChampionTMpET directional TOPO expression system)进行克隆表达,纯化获得N末端携带多聚组氨酸标签的重组HA,分子质量约78 ku.采用阳性血清经免疫印迹及ELISA分析重组HA的免疫反应性,结果表明重组HA能与H5N1亚型病毒抗血清发生特异性结合,具有良好的免疫反应性.
A pair of clone primers was designed and synthesized based on hemagglutinin（HA） gene sequences of known HSN1 subtype avian influenza viruses. HA gene were amplified from total RNA, which had been extracted from inactivated Yunnan HSN1 subtype virus positive clinical tissue sample, by reversing transcriptase-polymerase chain reaction using high proofreading polymerase （PyobestTM DNA Polymerase）, and was expressed using Invitrogen ChampionTM pET directional TOPO expression system. Recombinant HA con- taining polyhistidine （6xHis） tag in N-terminal is about 78 ku in size, which had been obtained and purified. Its immunoreactivity was analyzed by western blot and ELISA using positive serum. The results showed recombinant HA can bind to antiserum against HSN1 subtype virus with specificity and possesses good immunoreactivity.
出处
《云南大学学报（自然科学版）》
CAS
CSCD
北大核心
2007年第6期623-627,共5页
Journal of Yunnan University(Natural Sciences Edition)
基金
云南省科技攻关项目(2006NG-24)
昆明市科技重点资助项目(06N114134)
云南省后备人才基金资助项目(2005PY01-12)
关键词
禽流感病毒
H5N1亚型
血凝素
表达
avian influenza virus
H5 N 1 subtype
hemagglutinin
expression
