摘要
使用生化和免疫沉淀方法,对转基因CHO细胞B43系分泌的乙型肝炎(乙肝)病毒表面抗原(HBsAg)颗粒在SDS-PAGE上出现的30kD多肽,进行了分子组成的研究。首先用蛋白转移抗体酶染方法证明是特异性HBsAg,再用糖染色法证明是糖蛋白,以gp30命名之。观察到,随纯化产品在4℃保存时间的延长,gp30逐渐消失,说明gp30不够稳定。含gp30的样品经过胰酶消化后的电泳带形和血源HBsAg对照者基本相同。放射免疫沉淀试验发现细胞内的目的基因的翻译产物只有p23,其后逐步合成gp25、gp27及gp30。用衣霉素抑制或用内源性糖苷酶-F处理的样品,gp27的糖链消失,形成p23;而gp30的一个与gp27相同的糖链也消失,从而形成“gp27”,说明gp27及gp30中均有一个-N-连接的糖链。而在gp30中尚有另一种不受衣霉素和糖苷酶-F处理影响的、由-O-连接的糖基,由此形成“gp27”。分析工程细胞在合成、运输及分泌过程中逐步形成p23、gp25、gp27和gp30,后者是由p23双糖化而成。
As compared with plasma derived HBsAg, besides the two major poly-peptide bands p23 and gp27, an extra polypeptide band with molecular weight around 30kD was found in the SDS-PAGE of purified HBsAg from B43 cell line. Western blot showed that all polypeptide bands, including the 30kD polypeptide are constituents of the HBsAg particles. Carbohydrate staining of purified HBsAg indicated that 30kD polypeptide is also a glycoprotein, designated as gp30. The gp30 is unstable and disappeared after storing at 4℃ for two months. The result of the inhibition test by tunic-amycin and treatment with endo-glycosidase-F showed that gp27 contains a N-linkage polysaccharide chain, gp30 is a double glycosylated glycoprotein containing both N-linked and O-linked polysaccharide chain, gp27 and gp30 were formed from p23 during transportation and excretion.
出处
《病毒学报》
CAS
CSCD
北大核心
1990年第3期199-204,共6页
Chinese Journal of Virology
关键词
乙型肝炎病毒
表面抗原
蛋白糖化
Hepatitis B surface antigen Protein glycosylation Transgenic mammalian cells
