BCSC-1基因慢病毒干扰载体的构建与鉴定  

Construction and Identification of Lentiviral Vector Expressing shRNA for BCSC-1 Gene

作　　者：王洪伟[1] 刘义帅[1] 邸大琳[1] 魏兵[1] 吴国庆[1] 鞠吉雨[1] 

机构地区：[1]潍坊医学院免疫学教研室,山东潍坊261053

出　　处：《潍坊医学院学报》2014年第1期1-4,共4页Acta Academiae Medicinae Weifang

基　　金：山东省自然科学基金项目(课题编号:2009CM019)

摘　　要：目的构建BCSC-1基因慢病毒RNA干扰载体并感染人乳腺癌细胞MCF-7,实时荧光定量PCR(QPCR)检测干扰效果。方法 设计并合成两条针对人BCSC-1基因的干扰序列(BCSC-1 shRNA1和BCSC-1shRNA2)以及对照序列(NS),将其连接入载体PLKO.1-sp6-pgk-GFP中,得到两个干扰载体和一个阴性对照载体。通过菌落PCR、双酶切和DNA序列测定等方法对其进行鉴定。将干扰载体与辅助包装质粒Lipofectamine2000共转染HEK293T细胞包装慢病毒并收取病毒上清。所获慢病毒颗粒感染人乳腺癌细胞MCF-7,QPCR方法检测两种干扰载体对BCSC-1基因的抑制效率。结果 菌落PCR结果显示目的片段插入PLKO.1-sp6-pgk-GFP载体,DNA测序证实插入序列无误。QPCR结果显示两种干扰载体感染人乳腺癌细胞MCF-7后,BCSC-1 shRNA1组和BCSC-1 shRNA2组BCSC-1基因表达水平分别为0.34±0.046和0.63±0.043,较空白对照组和NS组均有明显下降(P<0.05),且BCSC-1 shRNA1抑制效果优于BCSC-1 shRNA2(P<0.05)。结论 成功构建了两种BCSC-1基因慢病毒干扰载体,两载体均能下调人乳腺癌细胞MCF-7中BCSC-1基因表达水平。Objective To construct lentiviral vectors expressing shRNA for BCSC-1 gene anti infect breast cancer cells MCF-7, real-time tluorescence quantitative PCR（ QPCR ） method was used to test the interference effect. Methods Two interfere sequences aiming to human BCSC-I gene（ BCSC-I shRNAI anti BCSC-I shRNA 2 ） and a control sequence（NS） were designed anti synthesized,inserting them in- to the shRNA expression vector PLKO. l-sp6-pgk-GFP. All the plasmids were proved c.orrect by colony PCR,restriction enzymes and I）NA se- qt, encing. The positive plasmid was transfected into HEK293T ceils with auxiliary plas,nids via Lipofectamine2000 to produce lentivirus and supernatant containing viruses was collected. The viruses infecting human breast carcinoma cell line MCF-7. QPCR method were used to de- tect the suppression efficiency. Results Colony PCR results indicated all the designed interfere sequences were successfully inserted into PL- KO. 1-sp6-pgk-GFP vector and DNA sequencing were correct. QPCR results indicated the BCSC-l gene expression levels in MCF-7 cells were decreased significantly in two interference vectors groups than NS group（P 〈 O. 05 vs NS group）. Moreover, the inhibition rate of BCSC-1 shRNA1 was higher than BCSC-1 shRNA1 （ P 〈 0.05 BCSC-1 shRNA1 vs BCSC-1 shRNA2 ）. Conclusion Two lentiviral vectors are suc- cessfully constructed. They can suppress the expression of BCSC-1 gene in MCF-7 ceils.

关 键 词：慢病毒载体 BCSC-1 基因 MCF-7细胞 实时荧光定量PCR 

分 类 号：R392-33[医药卫生—免疫学]