摘要
目的 获取具备免疫反应原性的庚型肝炎病毒ＮＳ３ 抗原。方法 从重组质粒Ｉｗｑ２利用ＰＣＲ 反应扩增出ＧＢＶＣ／ＨＧＶ ＮＳ３ 基因片段，克隆至表达载体ｐＰＲＯＥＸ ＨＴａ 后进行核苷酸序列测定，待鉴定正确后经ＩＰＴＧ 诱导重组工程菌，用ＳＤＳＰＡＧＥ 和Ｗｅｓｔｅｒｎ ｂｌｏｔ 对重组蛋白进行鉴定。结果 酶切鉴定和核苷酸序列测定结果表明成功地构建了重组工程菌ｐＨＴＮＳ３／ ＤＨ５α且克隆的基因片段为ＧＢＶＣ／ＨＧＶ ＮＳ３ 基因片段。ＳＤＳＰＡＧＥ 分析诱导表达产物发现在相对分子质量约４３ ８１０处有一条明显的蛋白表达带，占菌体总量的１７ ．７ ％ 。用ＮｉＮＴＡ 亲和层析柱快捷地获得了纯化的重组蛋白。Ｗｅｓｔｅｒｎ ｂｌｏｔ 鉴定发现重组蛋白可与ＧＢＶＣ／ＨＧＶ ＲＮＡ 阳性病人血清发生抗原抗体反应。结论 获得了具备免疫反应原性的ＧＢＶＣ／ＨＧＶ ＮＳ３ 抗原，该重组蛋白可用于ＧＢＶＣ／ＨＧＶ 感染的检测。
Objective To obtain immunoreactive GBV C/HGV NS3 protein. Methods GBV C/HGV NS3 gene was amplified from recombinant plasmid Iwq2 and cloned into pPROEX HTa expression vector. Nucleotide sequences of the cloned gene were determined and the cloned gene was identified as GBV C/HGV NS3 gene. The engineering E. coli pHTNS3/DH5α were induced by IPTG and the induced products were analyzed with SDS PAGE and Western blot. Results About a Mr 43?810 band was seen on the stained polyacrylamide gel which amounted to 17.7% of total bacteria proteins. The recombinant protein was purified with Ni NTA spin columns. Western blot demonstrated that reactions between recombinant protein and GBV C/HGV RNA positive sera occurred. Conclusion The recombinant NS3 protein has been obtained and can be used to detect GBV C/HGV infections.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
1999年第6期475-478,共4页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金
军队人才基金
