HBV A83点变异真核表达载体的构建及其在HepG2细胞中的表达  

Construction of eukaryotic expression vectors with A83 mutation in HBV gene

作　　者：林裕龙[1] 王战会[1] 侯金林[1] 孙剑[1] 阎丽[1] 骆抗先[1] 

机构地区：[1]第一军医大学南方医院传染料,广州510515

出　　处：《临床肝胆病杂志》2002年第1期20-21,共2页Journal of Clinical Hepatology

摘　　要：研究 Ａ８３点突变的生物学意义。采用分子生物学方法，设计引入 Ｋｐｎ　Ｉ和 Ｈｉｎｄ　ＩＩＩ酶切位点的引物扩增乙型肝炎病毒（ＨＢＶ）ａｙｗ亚型的ＰｃＰ１０标准株中的Ｐｒｅ—Ｃ／Ｃ片段（１８１４—２４５２），经Ｋｐｎ　Ｉ和Ｈｉｎｄ　ＩＩＩ双酶切后，在Ｔ４ＤＮＡ连接酶的作用下连于ＥＢ病毒真核表达载体，利用定点突变技术对连接载体进行１８９６点的定点诱变。经错配ＰＣＲ—ＲＦＬＰ和测序分析，确定突变的克隆。提取突变前后的质粒转染 ＨｅｐＧ２细胞。突变后在 Ｐｒｅ－Ｃ／Ｃ第２８位氨基酸形成终止密码，使ＨＢｅＡｇ合成终止，转染ＨｅｐＧ２细胞后未能检测到ＨＢｅＡｇ，而未突变的重组质粒则ＨＢｅＡｇ表达为强阳性。ＨＢＶ Ａ８３点突变真核表达载体的构建为体外研究该点突变对ＨＢｅＡｇ表达的影响以及ＨＢｅＡｇ阴性的乙型肝炎病毒感染的致病机理奠定基础。To study biological significance of mutation in HBV Pre C/C gene at nucleotide (nt)83. The primers with restriction sites of Kpn I and Hind III were designed to amplified Pre - C/C( 1 814 -2 452) from PcP10, which contains ayw subtype of HBV. After digested by Kpn I and Hind III, the PCR product was linked to EB viral eukarotic expression vector by T4 DNA linked enzyme. Then A83 variant was constructed by site - mutagenesis in vitro. The variant was identified by MPCR - RFLP and sequensing. Using the recombinant transfected HepG2 cell. The variant changes tryptophan into stop codon in twenty - eighth amino acid, which makes the formation of HBeAg stop. HBeAg can not be detected in medium from HepG2 cultured the variant and HBeAg was detected positive in medium from HexG2 cultured wild recombinant. The construction of A83 eukarotic expression vector will play an important pole in researching the effect on HBeAg expression and disease mechanism when A83 mutation in HBV signal peptide region.

关 键 词：定点突变 真核表达载体 转染 乙型肝炎病毒 致病机理 

分 类 号：R512.62[医药卫生—内科学]