乙型肝炎表面抗原S-蛋白糖化位点序列的人工变异及其哺乳动物细胞表达产物的性状研究  

EFFECT OF SITE DIRECTED REMOVAL OF N GLYCOSYLATION SITE OF HBsAg ON ITS BIOLOGICAL PROPERTIES

作　　者：刘文军[1] 杨芙蓉[1] 阮力[1] 任贵方[1] 朱既明[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》1997年第1期33-40,共8页Chinese Journal of Virology

基　　金：国家"八五"攻关资助

摘　　要：利用末端重叠ＰＣＲ法和定点突变技术，获得了去除Ｎ－连接多糖结构的乙肝表面抗原Ｓ突变基因，将乙肝表面抗原Ｓ－蛋白中结合Ｎ－连多糖结构序列Ａｓｎ－Ｘ－Ｔｈｒ中的第１４８苏氨酸的密码子ＡＣＴ，改变为编码甘氨酸的密码子ＧＧＴ。构建了该突变基因的表达载体并在ＣＨＯ细胞中表达，筛选到两株表达水平较高的细胞系。对其中的一株细胞系Ｃ４１的表达产物做了初步纯化和鉴定。纯化样品经ＳＤＳ－ＰＡＧＥ电泳分析，只含有２３ｋＤ一条蛋白带，而对照原基因ＣＨＯ细胞的表达产物则含有２３ｋＤ、２７ｋＤ和３０ｋＤ三条蛋白带，证实经人工修饰后其哺乳动物细胞表达产物不再含有糖基，纯化样品在电镜下可清楚地显示２２ｎｍ的球形颗粒。单克隆抗体反应谱分析发现，ＣＨＯ细胞表达的无糖ＨＢｓＡｇ与含糖ＨＢｓＡｇ的抗原表位存在明显差别。We used the overlap extension PCR to make a site-directed mutation on the HBV S gene,changing the threonine residue of the N-linked glycosylation sequence Asn-X-Thr to glycine.A pSV2-based plasmid containing the mutated S gene was constructed with the mutated S gene under the control of an SV40 early promoter and the dhfr - gene under the control of another SV40 early promoter.CHO dhfr - cells were transformed with the plasmid DNA and two cloned cell lines expressing HBsAg at high level were selected.SDS polyacrylamide gel electrophoresis showed that the purified product of the CHO C41 cell line have only a P23 band,while the control product from C28 cell line transformed by the non-mutated S gene showed three bands:P23,GP27 and GP30.This result proves that HBsAg produced by CHO C41 cell line has neither N-linked nor O-linked oligosaccharides.The purified HBsAg showed by electron microscopy were particles about 22nm in diameter,being similar to those found in C28 cell line.Five McAbs against different epitopesof the“a”determinant were used to analyze the epitopes of HBsAg from C41 cell line.The results showed that there are definite differences between HBsAg from C41 and C28 celll lines.

关 键 词：乙型肝炎 表面抗原 S-蛋白 糖化位点序列 变异 

分 类 号：R373.21[医药卫生—病原生物学]