期刊论文

中文

贵州医药

1000-744X

2019

43

5

675-678

10.3969/j.issn.1000-744X.2019.05.001

81673227:国家自然科学基金 81172712:国家自然科学基金 CX2018B628:湖南省研究生创新科研项目

本校为第一机构

目的 利用RNA干扰技术构建pYr-Lvsh-CAPNS1-shRNA载体,并在Hela细胞中鉴定.方法 针对目的基因人的CAPNS1基因(NM_001749),设计shRNA序列,以pYr-Lvsh为载体,构建pYr-Lvsh-CAPNS1-shRNA载体;干扰载体转染Hela细胞,利用荧光定量PCR检测CAPNS1 mRNA表达情况;荧光分光光度检测calpain活性;Western-blot检测相关蛋白含量.结果 结果发现,载体酶切及测序结果显示pYr-Lvsh-CAPNS1-shRNA载体构建成功;qPCR显示与对照组相比,shRNA组CAPNS1表达受到抑制,CAPNS1-sh的干扰效率为50.30％;calpain活性检测显示shRNA组calpain活性下降并且CAPNS1、calpain1和calpain2蛋白含量下降.结论 结果表明pYr-Lvsh-CAPNS1-s...

Objective To construct and identify the pYr-Lvsh-CAPNS1-shRAN vector by RNA interference in Hela cells. Methods According to the sequence of human CAPNS1 (NM_001749),shRNA was designed and pYr-Lvsh-CAPNS1-shRNAP was constructed by using Yr-Lvsh plasmid as a vector,and,then clones were digested with restriction endonuclease for identification and DNA sequencing.Then the plasmid was transfected into Hela cells.The expression of CAPNS1 mRNA was detected by qPCR,the activation of calpain was detected by Fluorescence spectrophotometry.The expression of related protein was detected by western-blot (...