我国河南株丁型肝炎病毒抗原在原核细胞中的高效表达及分泌  

HIGH- LEVEL EXPRESSION AND SECRETION OF CHINESE HENAN HEPATITIS DELTA ANTIGEN IN PROKARYOTIC CELLS

作　　者：刘善虑[1] 丛旭[1] 詹美云[1] 

机构地区：[1]中国预防医学科学院病毒学研究所

出　　处：《病毒学报》1995年第3期195-202,共8页Chinese Journal of Virology

基　　金：国家863高科技生物技术领域资助

摘　　要：将我国河南株丁型肝炎（丁肝）病毒抗原的编码基因，克隆到分泌性原核表达载体ｐＥＴ－１２ａ的Ｔ７启动子下游，构建了保留和去除终止子（Ｔｅｒｍｉｎａｔｏｒ）的两个表达载体ｐＥＴＤＡｇ和ｐＥＴＤＡｇ－１。将重组的表达质粒转化宿主菌ＢＬ２１，经ＩＰＴＧ诱导，成功地高效表达了丁肝病毒抗原（ＨＤＡｇ）。ＳＤＳ－ＰＡＧＥ及ｗｅｓｔｅｍｂｌｏｔ表明，分泌到细胞周间质和包涵体中的ＨＤＡｇ的分子量约为２７ｋＤ和２８ｋＤ。ＥＬＩＳＡ分析表明，分泌性ＨＤＡｇ的活性优于包涵体中的ＨＤＡｇ，分泌性ＨＤＡｇ的ＥＬＩＳＡ滴度高于１：３２０００比活性为１００００ＥＬＩＳＡ滴度／ｍｇ蛋白；包涵体ＨＤＡｇ的ＥＬＩＳＡ滴度可达１：２５６００，比活性为４５００ＥＬＩＳＡ滴度／ｍｇ蛋白。另外，利用本室建立的６株抗美国株ＨＤＡｇ单克隆抗体对该河南株重组丁肝病毒抗原（ｒＨＤＡｇ）进行了鉴定分析，表明它可与其中５株ＭｃＡｂ起特异性结合反应，而与另一株ＭｃＡｂ反应不好，推测可能与其ＨＤＡｇ的不同空间结构有关。wo expression plasmids pETDAg and pETDAg -1 ,which contain the gene of Chinese Henan hepatitis delta antigen were constructed under the T7 Promoter,with and without terminator resepectively .The host bacteria BL21 were transformed by the two recombinant plasmids and IPTG was used to induce HDAg expression .SDS-PAGE and Western blot showed that the molecualr weights of expressed HDAg in the periplasmic space and inclusion body were 27kD and 28kD respectively; ELISA tests showed that the antigentic activity of secreted HDAg in periplasmic space is 70% of the total ,its ELISA titer and specific activity are higher than 1:32,000 and 10,000 ELISA unit/mg of protein expressed. In addition ,several anti-HDV McAbs were used to analyse the possible epitopes of this Henan strain rHDAg and to compare with that of Sichuan strain . The result showed that one anti -HDV McAb ,McAb G12 ,can not bind rHDAg of this Henan strain ,though it reacts with rHDAg of Sichuan strain very well. This may be due to some differences in the tertiarystructuer of rHDAg between Henan and Sichuan .

关 键 词：丁型肝炎病毒 抗原 表达 分泌 

分 类 号：R373.21[医药卫生—病原生物学]