反义c-myc表达载体的构建及其瞬时表达对HepG2.2.15细胞的体外生物学效应

目的:构建反义RNA表达载体pcDNA3.1-as-myc,建立新型的c-myc反义RNA肝癌靶向性转移系统,研究瞬时表达对HepG2.2.15细胞的体外生物学效应。方法:XbaI和HindIII双酶切携带目的基因的质粒pcMYC和pcDNA3.1(-)真核表达载体,T4DNA连接酶连接,构建c-myc反义RNA表达载体。合成针对表皮生长因子受体(EGFR)的相应16肽GE7和流感病毒血凝素功能域的HA20寡肽,并与多聚赖氨酸连接,连接物与c-myc的反义RNA表达载体混合,构建新型c-myc反义RNA肝癌靶向性转移系统,即四元复合体。结果:成功构建反义RNA表达载体pcDNA3.1-as-myc,建立了as-myc四元复合体基因转移系统,经HepG2.2.15细胞系瞬时转染,证实c-myc蛋白的表达下降。结论:构建的反义表达载体pcDNA3.1-as-myc具有阻断c-myc表达的效应。

Construction of antisense c-myc expression vector and study on the biological effect of transient expression on HepG2.2.15 cell in vivo

Objective:To obtain recombinant expression vector pcDNA3.1with c-myc cDNA targeted to hepatocellular carcinoma and construct EGFR mediated hepatocarcinoma targeted gene transfer system-four element complex to study the biological effect of transient expression of antisense c-myc RNA on HepG2.2.15cell.Method:The plasmid pcMYC was excised using XbaI and HindIII and ligated into the same site of pcDNA3.1(-)excised by XbaI and HindIII in antisense directions.GE7and HA20were conjugated with polylysine to produce a conjugates that could in-teract with DNA to form the four element complex.Results:The recombinant pcDNA3.1-as-myc was successfully obtained by the molecular biological technique.After the transient transfection,ex-pression of c-myc protein was inhibited.Con clu sion:The recombinant expression vectors may lead to targeted-expression of c-myc on hepatoma and show tissue specificity and exclusiveness for hep-atoma.