衔接蛋白2基因siRNA表达载体的构建及鉴定

Construction and identification of siRNA expression vectors of adaptor protein 2 gene

目的:构建针对衔接蛋白2的μ2亚单位(AP2-μ2)的短链干涉RNA(si RNA)及其表达载体,转染细胞后观察其对AP2-μ2基因的干涉作用。方法:设计AP2-μ2靶向的发夹状si RNA,据此设计合成3条互补的寡核苷酸链,退火后分别连接到pSilencer3.1neo H1载体,转化扩增后进行序列测定。待转染细胞分为对照组、Scramble plasmid、AP2plasmid-1、AP2plasmid-2和AP2plasmid-3组。用脂质体转染法将3组si RNA重组质粒与阴性对照质粒(Scramble plasmid)转染大鼠心肌细胞株H9C2,通过RT-PCR法检测AP2-μ2基因表达水平的变化。结果:合成的6条寡核苷酸单链凝胶电泳显示多个电泳条带,分别退火后,产物经凝胶电泳显示形成单一条带。双链DNA模板与载体质粒连接后,用BamHⅠ和HindⅢ双酶切,凝胶电泳可见4300和66bp的酶切片段。DNA测序连接的核酸序列与设计序列一致,表明AP2-μ2基因的3条si RNA载体构建成功。RT-PCR检测,在AP2plasmid-1组,转染的H9C2细胞中AP2-μ2的基因表达水平较阴性对照组降低约96%。结论:成功地构建了针对AP2-μ2基因的3条si RNA载体,其中AP2plasmid-1转染细胞后可显著抑制AP2-μ2基因表达。

Objective To construct μ2 subunit of adaptor protein 2（AP2-μ2） small interfering RNA（siRNA） and expression vectors,and detect their silencing effects.Methods AP2-μ2 mRNA targeted hairpin siRNAs were devised and three oligonucleotide strands of DNA fragments encoding the above siRNAs were synthesized.After annealing of the complementary strands,the DNA fragments were cloned into pSilencer 3.1 neo H1 plasmid,followed by amplification in E.coli and DNA sequencing.The transfected cells were divided into five groups：control,scramble plasmid,AP2 plasmid-1,AP2 plasmid-2 and AP2 plasmid-3.Three groups of recombinant plasmids and the negative control siRNA plasmid（scramble plasmid） were transfected into rat cardiac cell line H9C2 by liposome transfection method,and the expression of AP2-μ2 mRNA was examined by RT-PCR.Results The synthesis of six single-stranded oligonucleotide showed multiple bands by gel electrophoresis.After annealing respectively,the product by gel electrophoresis showed a single band.After double-stranded DNA templates were connected with plasmid,they digested by restriction enzyme of BamHⅠ and Hind Ⅲ,the 4300 bp and 66 bp fragments were seen by gel electrophoresis.DNA sequencing analysis showed that the connecting sequence of nucleic acid sequences was same as the designed,indicating three AP2-μ2 siRNA vectors were successfully constructed.RT-PCR results showed that in AP2 plasmid-1 group,the AP2-μ2 gene expression level in transfected H9C2 cells reduced by 96% compared with negative control group.Conclusion Three kinds of AP2-μ2 siRNA vectors are successfully constructed,and AP2 plasmid-1 transfected cells can significantly inhibit the AP2-μ2 gene expression.