摘要
根据汉坦病毒Ｈ８２０５株ＮＰ基因的序列，设计一对引物，扩增ＮＰ前２９２个氨基酸多肽基因片段，克隆于表达载体ｐＧＥＸ３Ｘ，与载体中２６ｋＤ的谷胱苷肽巯基转移酶（ＧＳＴ）融合表达。ＳＤＳＰＡＧＥ显示表达产物（ＧＳＴＮＰ）主要以包涵体形式存在。Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ表明此融合蛋白有抗原性。包涵体经分离、洗涤、溶解后，Ｓｅｐｈａｒｏｓｅ６Ｂ层析纯化，用此融合蛋白作抗原，进行ＥＬＩＳＡ法检测临床ＨＦＲＳ病人标本的ＩｇＧ和ＩｇＭ，有很好的特异性和敏感性。有生物活性的汉坦病毒Ｈ８２０５ＮＰ的体外表达成功，为汉坦病毒基因工程抗原的大量制备奠定了基础，也为汉坦病毒的临床检测和流行病学调查提供了一种廉价、安全、可靠的抗原。
The S genome segment encoding the nucleocapsid protein (truncated) of Hantavirus H8205 was amplified by PCR and cloned into the expression vector pGEX 3X and expressed in E coli. SDS PAGE showed that the expressed fusion protein (GST NP) was mainly in the form of inclusion body. The expressed protein was used as diagnostic antigen in solid phase enzyme immunoassay. The assay was used to detect IgG and IgM antibody in sera of HFRS patients originated from different geographic regions of China. The results revealed highly sensitive and specific. The successful expression and application of recombinant nucleocapsid protein of Hantavirus provides cheap, safe and sensitive antigen for the diagnosis of HFRS in China.
出处
《中国病毒学》
CSCD
1999年第2期110-115,共6页
Virologica Sinica
关键词
汉坦病毒
核衣壳蛋白
ELISA
Hantavirus, Recombinant nucleocapsid protein, Enzyme linked immunosorbent assay (ELISA)
