摘要
目的构建抑制型pcDNA3/HA-moesin^(T558A)与激活型pcDNA3/HA-moesin^(T558D)基因突变体,并在人脐静脉内皮细胞(HUVECs)中验证。方法设计特定的moesin^(T558A)、moesin^(T558D)基因突变引物,以pcDNA3/HA-moesin为模板,采用逆向PCR技术进行体外定点突变,以丙氨酸、天冬氨酸取代苏氨酸,获得抑制型pcDNA3/HA-moesin^(T558A)和激活型pcDNA3/HA-moesin^(T558D)基因突变体并测序。体外培养HUVECs,随机分为对照1组、pcDNA3/HA空载体组、pcDNA3/HA-moesin组以及对照2组、pcDNA3/HA-moesin^(T558A)组、pcDNA3/HA-moesin^(T558D)组,pcDNA3/HA空载体组、pcDNA3/HA-moesin组、pcDNA3/HA-moesin^(T558A)组、pcDNA3/HA-moesin^(T558D)组分别转染pcDNA3/HA空载体、pcDNA3/HA-moesin、pcDNA3/HA-moesin^(T558A)、pcDNA3/HA-moesin^(T558D),对照1组与对照2组不予转染。转染24 h,收集细胞,采用实时荧光定量PCR法检测moesin mRNA表达,采用Western blotting法和免疫荧光法检测moesin蛋白表达。结果经测序,目的基因moesin^(T558A)、moesin^(T558D)序列完全正确,第558位苏氨酸分别突变为丙氨酸、天冬氨酸,抑制型pcDNA3/HA-moesin^(T558A)和激活型pcDNA3/HA-moesin^(T558D)基因突变体构建成功。pcDNA3/HA-moesin组moesin mRNA相对表达量显著高于对照1组和pcDNA3/HA空载体组(t分别为16.688、16.649,P均<0.05),而pcDNA3/HA空载体组与对照1组比较差异无统计学意义(t=1.925,P>0.05)。pcDNA3/HA-moesin^(T558D)组、pcDNA3/HA-moesin^(T558A)组moesin mRNA相对表达量均显著高于对照2组(t分别为13.809、9.260,P均<0.05),pcDNA3/HA-moesin^(T558D)组与pcDNA3/HA-moesin^(T558A)组比较差异无统计学意义(t=2.364,P>0.05)。Western blotting结果显示,pcDNA3/HA-moesin^(T558A)组和pcDNA3/HA-moesin^(T558D)组均能观察到HA-moesin蛋白表达,而对照1组未观察到HA-moesin蛋白表达。免疫荧光结果显示,pcDNA3/HA-moesin组、pcDNA3/HA-moesin^(T558A)组和pcDNA3/HA-moesin^(T558D)组均能观察到HA-moesin荧光,而对照2组未观察到HA-moesin荧光。�
Objective To construct suppressed pcDNA3/HA-moesin^(T558A) and activated pcDNA3/HA-moesin^(T558D) gene mutants and to verify their expression in human umbilical vein endothelial cells(HUVECs).Methods Specific moesin^(T558A) and moesin^(T558D) gene mutation primers were designed,and pcDNA3/HA-moesin was used as the template for in vitro targeted mutagenesis by reverse PCR,and threonine was replaced with alanine and aspartate to obtain suppressed pcDNA3/HA-moesin^(T558A) and activated pcDNA3/HA-moesin^(T558D) gene mutants and then they were sequenced.HUVECs cells were cultured in vitro and randomly divided into the control 1 group,pcDNA3/HA empty vector group,pcDNA3/HA-moesin group,and control 2 group,pcDNA3/HA-moesin^(T558A) group,and pcDNA3/HA-moesin^(T558D) group;the pcDNA3/HA empty vector group,pcDNA3/HA-moesin group,pcDNA3/HA-moesin^(T558A) group,and pcDNA3/HA-moesin^(T558D) group were transfected with pcDNA3/HA empty vector,pcDNA3/HA-moesin,pcDNA3/HA-moesin^(T558A),and pcDNA3/HA-moesin^(T558D),respectively,while the control 1 group and control 2 group were not transfected.The cells were transfected for 24 h.The moesin mRNA expression was detected by real-time fluorescence quantitative PCR,and the moesin protein expression was detected by Western blotting and immunofluorescence.Results After sequencing,the sequences of target genes moesin^(T558A) and moesin^(T558D) were completely correct,and the threonine at position 558 mutated into alanine and aspartate,respectively;the suppressed pcDNA3/HA-moesin^(T558A) and activated pcDNA3/HA-moesin^(T558D) mutants were successfully constructed.The relative expression of moesin mRNA in the pcDNA3/HA-moesin group was significantly higher than that in the control 1 group and the pcDNA3/HA empty vector group(t=16.688,16.649,both P<0.05),while there was no statistically significant difference between the pcDNA3/HA empty vector group and the control 1 group(t=1.925,P>0.05).The relative expression levels of moesin mRNA in the pcDNA3/HA-moesin^(T558D) and pcDNA3/HA-moesin^(T558
作者
刘红霞
陈力
王运林
罗卓章
黄巧冰
LIU Hongxia;CHEN Li;WANG Yunlin;LUO Zhuozhang;HUANG Qiaobing(Department of Endocrinology,Nanhai Hospital,Guangdong Provincial People's Hospital,Foshan 528000,China;不详)
出处
《山东医药》
CAS
2023年第13期6-9,14,共5页
Shandong Medical Journal
基金
国家自然科学基金资助项目(81870210)。
