[1]焉力方,李　鑫,姬文婕,等.小鼠P2X7受体基因短发夹RNA慢病毒载体构建与鉴定[J].新乡医学院学报,2017,34(5):351-355.[doi:10.7683/xxyxyxb.2017.05.002]
　YAN Li-fang,LI Xin,JI Wen-jie,et al.Construction and identification of lentiviral vector of short hairpin RNA of P2X7 receptor gene of the mouse[J].Journal of Xinxiang Medical University,2017,34(5):351-355.[doi:10.7683/xxyxyxb.2017.05.002]
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小鼠P2X7受体基因短发夹RNA慢病毒载体构建与鉴定
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
34
期数:
2017年5
页码:
351-355
栏目:
基础研究
出版日期:
2017-05-05

文章信息/Info

Title:
Construction and identification of lentiviral vector of short hairpin RNA of P2X7 receptor gene of the mouse
作者:
焉力方1; 李　鑫2; 姬文婕2; 周茂彬1; 李　琦2; 李玉明3; 周　欣3
（1.武警后勤学院，天津　300309；2.天津市心血管重塑与靶器官损伤重点实验室，天津　300162；3.武警后勤学院附属医院呼吸与重症医学科，天津　300162）
Author(s):
YAN Li-fang1; LI Xin2; JI Wen-jie2; ZHOU Mao-bin1; LI Qi2; LI Yu-ming3; ZHOU Xin3
(1.Logistics University of Chinese People′s Armed Police Force,Tianjin 300309,China;2.Tianjin Key Laboratory of Cardiovascular Remodeling and Target Organ Injury Institute of Cardiovacular Diease and Heart Center,Tianjin 300162,China;3.Department of Respiratory Medicine,the Affiliated Hospital of Logistics University of Chinese People′s Armed Police Force,Tianjin 300162,China)
关键词:
小鼠P2X7受体; RNA干扰; 慢病毒载体; RAW264.7细胞
Keywords:
P2X7 receptor; RNA interference; lentiviral vevtors; RAW264.7 cell
分类号:
Q782；R34
DOI:
10.7683/xxyxyxb.2017.05.002
文献标志码:
A
摘要:
目的　应用RNA干扰技术构建小鼠P2X7受体（P2X7R）基因重组慢病毒载体并鉴定。方法　对小鼠P2X7R mRNA分析，设计合成单链引物，经退火后形成双链寡核苷酸序列，放入经EcoR I和BamH I双酶切线性化的pLVX-shRNA-Puro慢病毒质粒载体中，连接构建慢病毒干扰载体pLVX-shRNA-P2X7R。筛选出阳性克隆，进行基因测序鉴定。测序验证正确后，与慢病毒包装辅助质粒psPAX2、pMD2.G共转染293T细胞，收集病毒上清。经浓缩后感染小鼠RAW264.7细胞，采用流式细胞术评价病毒滴度及感染效率；利用实时定量聚合酶链式反应（RT-PCR）和Western blot法鉴定P2X7R shRNA慢病毒的干扰效率。结果　测序证实，成功地构建了重组质粒pLVX-shRNA-P2X7R及小鼠P2X7R基因shRNA慢病毒载体。重组慢病毒的滴度为2×1010 TU·L-1，感染效率约为95%。干扰效率约为70%。结论　应用RNA干扰技术可成功构建小鼠P2X7R基因shRNA慢病毒载体。
Abstract:
Objective　To construct and identify the recombinant lentiviral vector of P2X7 receptor（P2X7R） gene in mouse.Methods　The single-stranded primer sequences were designed and synthesized based on mouse P2X7R gene sequence.After annealing,the double strand oligonucleotides was formed and then it was cloned into the lentiviral vector pLVX-shRNA-Puro which was digested by restriction endonuclease EcoR I and BamH I.The lentiviral interference vector of pLVX-shRNA-P2X7R was constructed.The positive clone was screened to identify the DNA sequence.After the DNA sequence was verified,the recombinant plasmid and two lentiviral packaging plasmids(psPAX2 and pMD2.G) were co-transfected into the 293T cells,and the virus supernatant was collected.The inspissated virus was used to infect RAW264.7 cells of mouse.The virus titer and infection efficiency of RAW264.7 cell was detected by flow cytometry;the interference efficiency of P2X7R shRNA lentivirus in RAW264.7 cell was measured by real-time polymerase chain reaction (RT-PCR) and Western blot.Results　The recombinant plasmid pLVX-shRNA-Puro-P2X7R and shRNA lentiviral vector of mouse P2X7R gene were constructed successfully.The titer of recombinant lentivirus was 2×1010 TU·L-1;the infection efficiency of RAW264.7 cell was 95%.The interference efficiency of P2X7 shRNA lentivirus in RAW264.7 cell was 70%.Conclusion　The shRNA lentiviral vector of P2X7R gene is constructed and identified successfully.

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更新日期/Last Update: 2017-05-05