人乙型肝炎病毒前表面抗原(preS)基因的突变及其在家蚕细胞中影响表达的研究

High-level Expression of Human HBV preS Gene in Silkworm ( Bombyx mori ) Cells by Mutation of the 5'-end Coding Sequence

为探讨人乙型肝炎病毒 (HBV)前表面抗原 (preS)基因的表达调控机理 ,以实现高效表达 ,利用PCR方法在克隆的 preS基因的第 2、3位密码子引入同义突变 ,消除存在于 5 '端编码区保守的反向重复序列 ,将 preS基因及其突变形式 (MpreS)分别重组到转移载体 pBM0 30 ,获得 pBM preS和pBM MpreS。将 pBM preS和 pBM MpreS分别与野生型家蚕核型多角体病毒 (BmNPV)DNA共转染家蚕培养细胞 (BmN) ,经空斑筛选和杂交证实 ,分别获得重组病毒rBmNPV preS和rBmNPV MpreS。RNA点杂交和ELISA结果表明 :虽然在rBmNPV preS和rBmNPV MpreS感染的BmN细胞内都转录了 preS基因 ,但仅后者表达出 preS蛋白 ,提示preS基因的表达与基因内部起始区的反向重复序列密切相关。

The surface proteins of hepatitis B virus (HBV) include S, M and L, which all possess immunogenic epitopes and therefore can be used as vaccines against HBV. High level expression of S and M has been demonstrated in extensive expression systems. However, native sequences of L (comprising preS+S) and preS proteins have not so far been reported to express efficiently. Evidences available showed that a conserved invert repeat in the 5′ end coding sequence accounted for the non expression in E. coli (Kim, et al., 1996). To investigate whether the invert repeat similarly influence expression efficiency in eukaryotic cells, we constructed a mutant gene with the invert repeat eliminated by synonymous mutation of ‘g 5gAggT 10 ’to‘g 5gTggA 10 ’, and evaluated expression efficiency of the native and mutant preS genes in baculovirus expression system. We found that the steady state levels of preS RNA were same for both native and mutant forms of pre S gene, however, only the mutant form expressed preS protein at a high level. These results indicated that the conserved invert repeat in the 5′end coding sequence of pre S gene played an crucial role in control of pre S gene expression in eukaryotic cells. Further, this control mechanism didn't work in transcription level but in translation level.