| 王美娟高美华△ 张蓓解西河王冰.人CD46 RNAi 逆转录病毒载体系统的构建与鉴定[J].现代生物医学进展英文版,2012,12(8):1414-1419. |
| 人CD46 RNAi 逆转录病毒载体系统的构建与鉴定 |
| Construction and Identification of Retro RNAi Systemof the Specific Silencing CD46 |
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| DOI： |
| 中文关键词: CD59 CD46 ShRNA 逆转录载体 |
| 英文关键词: CD59 ShRNA CD46 Retroviral vector |
| 基金项目:国家自然科学基金资助项目（3170893） |
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| 中文摘要: |
| 目的：构建并筛选携带针对CD46 基因的pSUPER retro RNAi 逆转录病毒载体，从而特异、有效地抑制人CD46mRNA 水平。
方法：利用DNA 重组技术，将2 条60nt 能转录产生靶向CD46 小发夹RNA（shRNA）的寡核苷酸序列，定向克隆入逆转录病毒载
体pSUPER retro，并转化大肠杆菌JM109。结果：重组载体经PCR 及限制性内切酶酶切鉴定初步成功后送测序，结果表明序列正
确。结论：特异性沉默CD46 基因的pSUPER retro RNAi 逆转录病毒载体构建成功，为后续转染Jurkat 细胞，研究CD46 在T 细胞
的信号转导中的作用奠定了基础，对进一步研究T 细胞相关疾病及开展细胞免疫缺陷的治疗方面提供了新的思路与方向。 |
| 英文摘要: |
| Objective: To construct recombinant retrovirus encoding shRNA targeted human CD46 and identify its function.
Methods: shRNA was synthesized, annealed and inserted into pSUPER plasmids digested with BglⅡand HindⅢ to construct the recombinant
pSUPER RNAi plasmids(pSUPER-CD46). Recombinant pSUPER-CD46 plasmid was identified by enzyme digestion and sequencing
analysis. The plasmids were transfected into Jurkat cells in vitro and the inhibitory efficiency of target genes expression was observed
with RT- PCR and Western blot. Recombinant pSUPER-CD46 vector was identified correct by enzyme digestion and sequencing
analysis. Result: The siRNA eukaryotic expression vectors constructed targeting on CD46 could reduce the expressions of target genes
and it might be able to use for the exploration of new anti-fibrosis drugs genetically. Conclusion: The eukaryotic expression vectors were
constructed successfully. Future work could investigate the relationship between CD59 and CD46 in T cell signal transduction. |
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