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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。
In order to explore the mechanism of expression regulation of preS gene of human hepatitis B virus (HBV) in order to achieve high expression, synonymous mutations were introduced into codons 2 and 3 of preS gene by PCR to eliminate the existence The conserved inverted repeat sequences of the 5 ’end coding region were obtained. The preS gene and its mutated form (MpreS) were recombined into the transfer vector pBM0 30 to obtain pBM preS and pBM MpreS, respectively. The BmN was co-transfected with pBM preS and pBM MpreS respectively with wild-type BmNPV DNA. The recombinant viruses rBmNPV preS and rBmNPV MpreS were obtained respectively by plaque screening and hybridization. The result of RNA dot blot and ELISA showed that although the preS gene was transcribed in BmN cells infected with rBmNPV preS and rBmNPV MpreS, only the latter expressed preS protein, which indicated that the expression of preS gene was reverse to that of the internal start region The sequence is closely related.