乙型肝炎病毒e抗原的纯化及其在人血清抗体检测中的应用

Purification of hepatitis B virus e antigen and its application in detection of human serum antibody

目的方法利用ＺｈｏｕＪｉａｎ教授提供的重组质粒ＤＮＡｐＢＶ２２０／ｅＡｇ，转染ＤＨ５α细胞，经３０℃培养３小时，４２℃培养６小时温度诱导后，提取乙型肝炎病毒ｅ抗原（ＨＢｅＡｇ）。经ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＦａｓｔＦｌｏｗ层折纯化后免疫打点分析，收集阳性蛋白。经ＳＤＳ－ＰＡＧＥ电泳，考马斯亮蓝Ｇ２５０染色，显示为单一的蛋白带，用免疫印迹法（Ｗｅｓｔｅｒｎｂｌｏｔ）和免疫打点（ｌｍｍｕｎｏｄｏｔ）法，确定表达产物的特异性。将纯化的ＨＧｅＡｇ用酶联免疫吸附试验（ＥＬＩＳＡ）和免疫条。方法基检测人血清中的相关抗体。结果经４９例乙型肝炎病毒ｅ抗体（ＨＢｅＡｂ）阳性病人血清检验证实了其特异性。结论免疫条方法较ＥＬＩＳＡ方法更敏感、特异。

A recombinant plasmid DNA pBV220/eAg provided by professor Zhou Jian was transferred into DH5 α celes.The effectively expressed strain was harvested after cloning.It was then incubated in 30℃ for 3 hrs and 42℃ for 6 hrs,the HBeAg protein was induced.After purification of the protein by DEAE-Sepharose Fast Flow chromatography,the purified protein was analyzed by SDS-PAGE electrophoresis and Coomassic brilliant blue R250 stain and only one single protein band was seen, Its specificity was demonstrated by Western-blot and Immunodot. 49 HBeAb positive sera of hepatitis B patients could be specifi cally detected with the purified HBeAg by ELISA and immunostrip assay,but the latter was more sensitive and specific.