【摘要】 目的构建编码大鼠AT1-R mRNA的shRNA真核表达载体质粒,为利用基因沉默技术从转录后水平进行高血压治疗的研究做准备。方法根据大鼠AT1-R mRNA序列设计并合成shRNA寡核苷酸片段,退火形成双链并克隆进入载体pGenesil-1,并进行酶切鉴定和测序。结果酶切证明构建的shRNA已插入载体,经测序证明与设计的相同,获得大鼠AT1受体的shRNA表达载体质粒。结论构建的编码大鼠AT1-R mRNA的shRNA真核表达载体可进入哺乳细胞内表达短发夹RNA,经Dicer酶裂解成siRNA并降解靶基因mRNA,而达到基因干扰的作用。为利用其进行自发性高血压大鼠的降压治疗研究奠定了基础。更多还原

【Abstract】 Objective To construct the expression vector of shRNA targeted to the rat angiotensin Ⅱ receptor gene for antihypertensive therapy in spontaneous hypertensive rat(SHR) in post-transcriptional regulation.Methods The sense and antisense RNA oligonucleotide strands targeting angiotensin Ⅱ receptor mRNA(Accession No.NM-030985) were synthesized individually according to the sequence of the rat angiotensin Ⅱ receptor.For preparation of duplexes,sense-and antisense-stranded oligonucleotides(20 μmol/L each) were mixed together in annealing buffer(final concentration of 50 mmol/L NaCl and Tris-HCl,pH 8.0),incubating in 94 ℃ for 1 min,and cooling slowly at room temperature.Then the annealed duplexes were cloned into the BamHⅠand Hind Ⅲ sites of the pGenesil-1 vector,generating the construct pGenesil-1-shRNA.Results The cloned shRNA construct which digested with Pst I and Sal I enzymes was inserted into the pGenesil-1 vector and verified with restriction enzymes,and the inserted sequences were verified by DNA sequencing,thus the plasmid pGenesil-1-shRNA vector was gained.Conclusions Plasmid-based shRNA expression systems targeted against the rat angiotensin Ⅱ receptor gene are generated successfully,the shRNAs with a 22nt stem and a short loop are cleaved into small interfering dsRNA(siRNA) by the Dicer.The siRNA enables the effective silencing of gene expression by presenting various siRNA sequences to the target mRNA and will be useful for the antihypertensive research in SHR(spontaneous hypertension rats).更多还原