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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
文章发布日期:2009年10月21日  来源:  作者:  点击次数:1505次  下载次数:624次

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【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。
【Abstract】: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 reg
【关键字】:定点突变; 真核表达载体; 转染
【Key words】:site - mutagenesis; eukaryotic expression vector; transfection
【引证本文】:

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