RAB5A蛋白G81R位点突变对其功能的影响  

The Effects of Mutant Site G81R of RAB5A on RAB5A Function

作　　者：宋春娇[1] 吕冰洁[1] 史忠诚[1] 于旸[1] 李钰[1] 李璞[1] 

机构地区：[1]哈尔滨医科大学遗传学和细胞生物学教研室,哈尔滨150086

出　　处：《Acta Genetica Sinica》2003年第10期967-972,共6页

基　　金：国家自然科学基金资助项目 (No .3 9970 3 96;No .3 0 170 5 16);国家自然基金国际合作项目 (No .3 0 2 13 0 2 3 );黑龙江省自然科学基金(D980 9)~~

摘　　要：为探讨G81R位点突变对RAB5A蛋白功能的影响 ,将RAB5AG81R突变体和正常RAB5A反义RNA分别插入pcDNA3 1 V5 His TOPO真核表达载体 ,并转染到Anip973细胞。用Westernblot方法检测稳定转染后细胞RAB5A蛋白的表达水平 ,通过血清饥饿法使Anip973和转染后的细胞同步化 ,停滞于G0 ～G1 期 ,再恢复血清培养使细胞从G0 ～G1 期释放 ,用流式细胞分析仪分析细胞周期各时相细胞百分比。结果显示RAB5AG81R突变体的反义RNA可完全封闭Anip973细胞中RAB5A的表达 ,正常RAB5A反义RNA部分封闭RAB5A表达。并且 ,Anip973细胞周期的长短与RAB5A的表达程度成反比。RAB5AG81R反义RNA能够有效地阻断Anip973细胞中RAB5A蛋白的表达。To study the effects of mutant site G81R of RAB5A ,t wo antisense RNA of RAB5A G81R and RAB5A were inserted into pcDNA3.1/V5-His TOPO expression vector,and transfected into Anip973,respectively,then detecte d the protein expressional level of RAB5A by Western blot.Finally,we synchronize d Anip973 and the two transfected cells at G 0/G 1,released cells at G 0/G 1 by culture medium containing FBS,and sequentally analyzed the percentage of cel ls at G 0/G 1,S and G 2/M by FCM.The expression of RAB5A may be completely bl ocked by the antisense RNA of RAB5A G81R,while the antisense RNA of RAB5A partia lly blocked expression of RAB5A.Futhermore,cell cycle of Anip973 was reciprocity to the protein expressional level of RAB5A.The antisense RNA of RAB5A G81R eff ectively blocked the expression of RAB5A in Anip973.Cell cycle was lengthened by blocking or reducing expression of RAB5A G81R.

关 键 词：反义RNA RAB5A 突变 

分 类 号：Q754[生物学—分子生物学]