大鼠4-1BB基因shRNA表达质粒的构建、鉴定和筛选  

Establishment and identification of 4 -1BB shRNA expressing plasmid

作　　者：侍阳[1] 宋庆伟[1] 于生才[1] 周晓俊[2] 殷骏[2] 钱海鑫[2] 

机构地区：[1]徐州医学院附属医院普外科,江苏徐州221002 [2]苏州大学附属第一医院普外科,江苏苏州215006

出　　处：《徐州医学院学报》2013年第2期71-75,共5页Acta Academiae Medicinae Xuzhou

基　　金：江苏省自然科学基金（BK2007057）;徐州市科技计划项目（XM098092）;徐州医学院院长专项基金（09KJZ09）

摘　　要：目的为研究小干扰RNA（siRNA）在移植免疫、肿瘤防治等方面的应用，构建针对大鼠T细胞4-1BB基因编码区的短发夹RNA（shRNA）表达质粒。方法根据RNA干扰（RNAi）作用原理，设计、合成4条T细胞4-1BB基因的shRNA（shRNA441、shRNA540、shRNA621、shRNA758）DNA片段，插入质粒pGPU6，构建4个编码目的shRNA的表达质粒（p441、p540、p621、p758），酶切及测序鉴定。设对照组，利用电穿孔法在体外将目的基因片段导入BN大鼠T细胞，48h后，逆转录-聚合酶链反应（RT-PCR）和流式细胞术（FCM）检测其对T细胞4-1BB基因表达的抑制作用，并筛选、确定最佳效果的干扰质粒。结果酶切及测序证实成功构建重组干扰质粒v441、p540、p621和p758，转染T细胞后，4-1BB相对表达量分别为（33．4±2．3）％、（53．0±2．3）％、（62．1±3．1）％、（40．9±1．5）％，对照组4-1BB相对表达量为（67．2±1．5）％。其中p441沉默效果最佳（P〈0．01）。结论siRNA具有抑制活化T细胞4-1BB基因表达的作用，干涉靶点具有选择性。Objective To construct 4 - 1BB specific short hairpin RNA （shRNA） expression plasmids to meet the requisition for further study. Methods According to the mechanism of RNA interference （ RNAi ）, 4 expression se- quences of shRNA target to rat lymphocyte 4 - 1BB were designed （shRNA441, shRNA540, shRNA621, shRNA758）, and inserted into expression vector pGPU6. The recombined plasmid vectors （p441, p540, 13621, p758） were identified by restriction enzyme analysis and sequencing, then, transfected into rat lymphocytes with electroporation. The changes of 4- 1BB mRNA level and gene expression were determined by semi -quantitative reverse transcription -polymerase chain reaction （ RT - PCR） and flow cytometry （FCM） at 48 h after transfeetion. Meanwhile, one of the recombined plasmids was judged optimization for following experiments. Results The interferential plasmid p441, p540, p621, and p758 were successfully constructed. Relative expression of 4 - 1BB on activated T lymphocytes subjected to those plasmid were （33.4±2.3）%, （53.0±2.3）%, （62.1 ±3.1）% and （40.9±1.5）%, respectively, while （67.2±1.5）% in control group. Silencing effect of 13441 was the best （P 〈 0.01 ）. Conclusions siRNA can decrease the expression of 4 - 1BB mRNA and protein in rat T lymphocytes. Electroporation technique is an effective method for transfecting plasmid to some difficult - transfeetion cells, such as T lymphocytes.

关 键 词：4-1BB(CD137) 小干扰RNA 基因沉默 

分 类 号：Q52[生物学—生物化学]