人热休克蛋白90β基因沉默质粒的构建及转染效率的观察

Construction of si-RNA synthesis plasmids targeting human Hsp90β gene and observation of their transfection efficiencies

目的:构建能表达小发夹RNA,从而特异、有效抑制人Hsp90βmRNA水平的质粒,比较该质粒对不同细胞株的转染效率。方法:合成3条对应于靶基因3个区域的64nt寡聚核苷酸,插入pSUPEREGFP1质粒,用DH5α细菌扩增,用酶切和测序法鉴定。以绿色荧光蛋白表达为标记,比较不同比例脂质体和质粒条件下,HepG2、HUVEC、HeK2933种细胞株的转染效率。结果:构建的pSuper-Hsp90β1、pSuper-Hsp90β2、pSuper-Hsp90β33个重组沉默质粒,插入片段碱基完全正确。在相同条件下,所构建的质粒对HeK293细胞转染效率最高,HepG2细胞最低。结论:利用pSUPER质粒可成功构建人Hsp90β基因沉默质粒,该质粒对细胞株的转染效率可因细胞的不同而有显著差异。

Objective：To construct the plasmid that can direct the synthesis of si-RNA like transcripts to specifically and effectively in hibit the mRNA level of human Hsp90βgene, and to compare the transfection efficiencies of the plasmids in different cell strains. Methods： Three 64 nt oligos corresponding to different regions in the target gene are chemically synthesized and annealed, then inserted into pSUPER EGFP1,proliferated by DH5a, and determined by endodigestion and sequencing. Three strains, HepG2、HUVEC and HeK293, were cultured. We then transfected the plasmids into the cells under different ratio of plasmid and Lipofectamine,and compared the transfection efficiencies of them by detection of EGFP Fluoresence. Results：The presence of positive recombinant clones was verified by double-digestion and sequencing. The bases inserted into the pasmids were correct.And the positive colonies were named as pSuper-Hsp90β1、pSupor-Hsp90β2 and pSuper-Hsp90β3, and the same condition,the constructed plasmid had the highest transfection efficiency in HeK293, and the lowest in HepG2 cells. Conclusions：The si-RNA synthesis plasmids targeting Hsp90β could be successfully constructed by using psupor plasmids, and the difference of their transfection efficiencies in different cells was significant.