小鼠P2X_7受体基因短发夹RNA慢病毒载体构建与鉴定
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摘要
目的应用RNA干扰技术构建小鼠P2X_7受体(P2X_7R)基因重组慢病毒载体并鉴定。方法对小鼠P2X_7R 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-P2X_7R。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P2X_7R 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-P2X_7R及小鼠P2X_7R基因shRNA慢病毒载体。重组慢病毒的滴度为2×1010TU·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P2X_7R基因shRNA慢病毒载体。
Objective To construct and identify the recombinant lentiviral vector of P2X_7receptor( P2X_7R) gene in mouse. Methods The single-stranded primer sequences were designed and synthesized based on mouse P2X_7 R gene sequence. After annealing,the double strand oligonucleotides was formed and then it was cloned into the lentiviral vector pLVXshRNA-Puro which was digested by restriction endonuclease EcoR Ⅰ and BamH Ⅰ. The lentiviral interference vector of pLVXshRNA-P2X_7 R 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 the293 T 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 P2X_7 R 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-P2X_7 R and shRNA lentiviral vector of mouse P2X_7 R gene were constructed successfully. The titer of recombinant lentivirus was 2 × 1010TU·L~(-1); the infection efficiency of RAW264. 7 cell was 95%.The interference efficiency of P2X_7 shRNA lentivirus in RAW264. 7 cell was 70%. Conclusion The shRNA lentiviral vector of P2X_7 R gene is constructed and identified successfully.
Objective To construct and identify the recombinant lentiviral vector of P2X_7receptor( P2X_7R) gene in mouse. Methods The single-stranded primer sequences were designed and synthesized based on mouse P2X_7 R gene sequence. After annealing,the double strand oligonucleotides was formed and then it was cloned into the lentiviral vector pLVXshRNA-Puro which was digested by restriction endonuclease EcoR Ⅰ and BamH Ⅰ. The lentiviral interference vector of pLVXshRNA-P2X_7 R 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 the293 T 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 P2X_7 R 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-P2X_7 R and shRNA lentiviral vector of mouse P2X_7 R gene were constructed successfully. The titer of recombinant lentivirus was 2 × 1010TU·L~(-1); the infection efficiency of RAW264. 7 cell was 95%.The interference efficiency of P2X_7 shRNA lentivirus in RAW264. 7 cell was 70%. Conclusion The shRNA lentiviral vector of P2X_7 R gene is constructed and identified successfully.
引文
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[19]RAMIREZ A,RATHINAM V,FITZGERALD K A,et al.Defective pro-IL-1beta responses in macrophages from aged mice[J].Immun Ageing,2012,9(1):27.
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[21]谢遂亮,杨达胜,毕凌云,等.小鼠脂肪源间充质干细胞的培养鉴定及增强绿色荧光蛋白-慢病毒载体转染[J].新乡医学院学报,2013,30(11):865-870.
[22]LOIS C,HONG E J,PEASE S,et al.Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors[J].Science,2002,295(5556):868-872.
[2]AMADIO S,D'AMBROSI N,CAVALIERE F,et al.P2 receptor modulation and cytotoxic function in cultured cns neurons[J].Neuropharmacology,2002,42(4):489-501.
[3]BEAMER E,FISCHER W,ENGEL T.The atp-gated P2X7receptor as a target for the treatment of drug-resistant epilepsy[J].Front Neurosci,2017,11:21.
[4]GALAM L,RAJAN A,FAILLA A,et al.Deletion of P2X7attenuates hyperoxia-induced acute lung injury via inflammasome suppression[J].Am J Physiol Lung Cell Mol Physiol,2016,310(6):572-581.
[5]ROGER S,JELASSI B,COUILLIN I,et al.Understanding the roles of the P2X7receptor in solid tumour progression and therapeutic perspectives[J].Biochim Biophys Acta,2015,1848(10 Pt B):2584-2602.
[6]DEL PUERTO A,FRONZAROLI-MOLINIERES L,PEREZ-ALVAREZ M J,et al.Atp-P2X7receptor modulates axon initial segment composition and function in physiological conditions and brain injury[J].Cereb Cortex,2015,25(8):2282-2294.
[7]ZHANG J,LI X,GAO Y,et al.Effects of puerarin on the inflammatory role of burn-related procedural pain mediated by P2X(7)receptors[J].Burns,2013,39(4):610-618.
[8]RISSIEK B,HAAG F,BOYER O,et al.P2X7on mouse T cells:one channel,many functions[J].Front Immunol,2015,6:204.
[9]TOKI Y,TAKENOUCHI T,HARADA H,et al.Extracellular atp induces P2X7receptor activation in mouse kupffer cells,leading to release of IL-1beta,HMGB1,and PGE2,decreased MHC class I expression and necrotic cell death[J].Biochem Biophys Res Commun,2015,458(4):771-776.
[10]苏程程,张译丹,马永强,等.干扰P2X7R基因对RAW264.7细胞增殖和吞噬的影响[J].中国病理生理杂志,2015,31(11):2065-2069.
[11]ZHANG K,LIU J,YOU X,et al.P2X7as a new target for chrysophanol to treat lipopolysaccharide-induced depression in mice[J].Neurosci Lett,2016,613:60-65.
[12]张艳芬,许重洁,杨保胜,等.环磷酰胺对小鼠腹腔巨噬细胞功能的影响[J].新乡医学院学报,2008,25(4):366-368.
[13]许莉,李东升,董碧麟,等.白色念珠菌经组织蛋白酶b途径诱导小鼠腹腔巨噬细胞nlrp3炎性体表达[J].新乡医学院学报,2014,31(1):1-4.
[14]GORDON S,MARTINEZ F O.Alternative activation of macrophages:mechanism and functions[J].Immunity,2010,32(5):593-604.
[15]杨志勇.胃癌中肿瘤相关巨噬细胞的分布及其与患者预后的关系[J].新乡医学院学报,2012,29(1):52-54.
[16]XU K,HARRISON R E.Down-regulation of stathmin is required for the phenotypic changes and classical activation of macrophages[J].J Biol Chem,2015,290(31):19245-19260.
[17]KOZICKY L K,ZHAO Z Y,MENZIES S C,et al.Intravenous immunoglobulin skews macrophages to an anti-inflammatory,IL-10-producing activation state[J].J Leukoc Biol,2015,98(6):983-994.
[18]MEHTA N,KAUR M,SINGH M,et al.Purinergic receptor P2X(7):a novel target for anti-inflammatory therapy[J].Bioorg Med Chem,2014,22(1):54-88.
[19]RAMIREZ A,RATHINAM V,FITZGERALD K A,et al.Defective pro-IL-1beta responses in macrophages from aged mice[J].Immun Ageing,2012,9(1):27.
[20]ZANIN R F,BERGAMIN L S,MORRONE F B,et al.Pathological concentrations of homocysteine increases IL-1beta production in macrophages in a P2X7,NF-κB,and ERK-dependent manner[J].Purinergic Signal,2015,11(4):463-470.
[21]谢遂亮,杨达胜,毕凌云,等.小鼠脂肪源间充质干细胞的培养鉴定及增强绿色荧光蛋白-慢病毒载体转染[J].新乡医学院学报,2013,30(11):865-870.
[22]LOIS C,HONG E J,PEASE S,et al.Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors[J].Science,2002,295(5556):868-872.