STRUCTURE IN THE PRECORE REGION OF HEPATITIS B CORE GENE AFFECTING ITS EXPRESSION IN E.coil

Restriction fragments of HBV-DNA, cleaved by endonuclease HhaI,containing HBcAg gene were trimmed by BAL-31 exonuclease to remove different lengths of the precore sequence.They were inserted into plasmid pUR222 at EcoRI site through synthetic linker ligation. Transformants in E.coli BMH7118 showing different levels of HBcAg gene expression were screened and analyzed for their nucleotide sequences in the junction region both by Maxam and Gilbert's chemical degradation method and by M13 chain termination method. Results of sequence analysis of different transformants revealed a partial palindromic (loop and stem) structure, at -7 to -35 nucleotide with regard to ATG of the HBcAg gene as position +1, which has dramatic effect on the level of expression of the inserted gene using the same promoter,SD sequence and identical N-terminus.The amount of HBcAg synthesized differed from 9% in the high expressing plasmid to less than 0.01% of the total cell proteins in the low expressing transformants.The findings w     

乙型肝炎病毒核心抗原基因结构对其在大肠杆菌中表达的影响

本文将含乙型肝炎(以下简称乙肝)病毒(HBV)核心抗原(HBcAg)基因的1kbHhaI酶切HBV·DNA片段用BAL-31外切酶切除两端非编码区,连接EcoRI合成接头后插入到载体pUR222的EcoRI位点,转化受体菌后筛到一系列不同HBcAg表达水平的重组体。分析高表达和低表达重组体工作中,发现在同一乳糖启动子、核糖体结合部位(SD序列)和Lac Z基因N端第5氨基酸后连接的HBcAg基因,由于在ATG5′端上游—7至—35核苷酸区有一呈部分迥文的序列,当转录成mRNA时可形成一环茎二级结构,是否用BAL—31酶切除此结构对表达水平影响极大。高表达重组体HBcAg产量可占菌体总蛋白9％,低表达的只为其千分之一。本文并对此现象在原核细胞中表达真核基因的意义进行了讨论。