构建Cx43特异性shRNA真核表达载体及体外干扰效率的鉴定  

Construct an shRNA Expression Vector Targeting Cx43 and Identify the Efficiency of RNA Interference in Vitro

作　　者：郑翠红[1] 黄光英[1] 张明敏[2] 王伟[3] 

机构地区：[1]华中科技大学同济医学院附属同济医院中西医结合研究所,武汉430030 [2]华中科技大学同济医学院附属同济医院中西医结合科,武汉430030 [3]华中科技大学同济医学院附属同济医院神经科,武汉430030

出　　处：《中西医结合研究》2009年第1期1-5,共5页Research of Integrated Traditional Chinese and Western Medicine

基　　金：国家自然科学基金重大研究计划(No.90209009);国家重点基础研究发展计划(973;No.2006CB504502)

摘　　要：目的本研究前期研究结果表明Cx43与穴位、经脉之间存在着密切联系,为了进一步研究Cx43在针刺活动中的作用,我们应用RNA干扰技术首先构建针对Cx43基因的特异性shRNA(smallhairpinRNA)真核表达载体,并转染体外培养的NIH/3T3细胞,观察其对Cx43基因的沉默效果,从而挑选出一条能有效沉默Cx43表达的shRNA,为进一步在体研究作准备。方法针对大、小鼠Cx43mRNA同源序列的两个靶点,体外合成两对互补的寡核苷酸链。经退火连接形成双链,插入到PGenesil-1真核表达载体。经酶切鉴定及测序等步骤后,将构建好的表达载体[分别命名为P-Cx43-shRNA(1)、P-Cx43-shRNA(2)]分别转染NIH/3T3细胞,G418筛选后应用免疫荧光、Western blot等方法观察P-Cx43-shR-NA对Cx43蛋白水平的影响。结果酶切鉴定及测序结果表明,成功构建了两个针对Cx43基因的shRNA真核表达载体及对照质粒(P-con-shRNA);Western blot结果说明两个Cx43特异性shRNA真核表达载体分别使Cx43蛋白水平下降了73.5%(P<0.01)和10.8%(P>0.05)。阴性对照质粒(P-con-shRNA)对NIH/3T3细胞Cx43蛋白水平无明显影响。结论重组质粒P-Cx43-shRNA(1)能较好的特异性抑制Cx43基因在NIH/3T3细胞中的表达,为进一步在体研究提供了实验依据。Objective Our previous studies showed that there were close relationships between Cx43 and acupoints and meridians. In order to further investigate the effect of Cx43 in acupuncture treatments, we use RNA interference technology to construct an shRNA expression vectors targeting Cx43 and identify the efficiency of RNA interference in NIH/3T3 cell lines for the further use in vivo. Methods Aiming directly at the two targets of Cx43 mRNA sequence of rat and mouse homology region,we synthetized two pair complementary oligonucleotide strands in vitro. Double strands were formed after annealing,which were then inserted into Pgenesil-1 plasmid expression vector. After identification by enzyme cutting and sequencing, the recombined plasmids named P-Cx43-shRNA（ 1 ） , P-Cx43-shRNA（2） and P-con-shRNA were transfected into the NIH/3T3 cells. Immunofluorescence and Western blot assays were used to detect the effect of the plasmid on the protein level of Cx43 after being screened by G418. Results The results of enzyme cutting and sequencing showed that we successfully constructed two shRNA expression vectors targeting Cx43 and a control expression vector for rat and mouse. And the Cx43 protein level was decreased by 73.5% （P 〈 0.01 ）and 10.8% accordingly. The Cx43 protein level was not influenced by the transfection of P-con-shRNA. Conclusion The plasmid P-Cx43-shRNA（ 1 ） can specificly silence better the expression of Cx43 in the NIH/3T3 cells ,which offers an experimental evidence for further in vivo investigation.

关 键 词：RNAI CX43 SHRNA 针刺 

分 类 号：R341[医药卫生—基础医学]