RNA干扰端粒酶反转录酶基因慢病毒表达载体的构建及鉴定  

Construction and identification of recombinant lentivirus expressing small interfering RNA against human telomerase reverse transcriptase gene

作　　者：宋扬[1] 徐韬[1] 杨明坤[2] 王国旗[1] 张恩丰 盛伟斌[1] 

机构地区：[1]新疆医科大学第一附属医院脊柱外科,新疆维吾尔自治区乌鲁木齐市830054 [2]四川省巴中市中心医院骨科,四川省巴中市636600

出　　处：《中国组织工程研究》2014年第11期1724-1729,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金资助项目(81060106)~~

摘　　要：背景:端粒酶反转录酶(telomerase reverse transcriptase,TERT)对端粒酶活化起重要作用,但利用构建针对其基因的慢病毒抑制其在脊髓星形胶质细胞中的表达却少有报道。目的:构建用于RNA干扰的靶向大鼠脊髓源星形胶质细胞端粒酶反转录酶基因的慢病毒载体,并观察其对端粒酶反转录酶表达的抑制作用。方法:通过设计合成出shRNA-TERT序列,经聚合酶链反应扩增后定向连接到pLentilox3.7U6载体上构建重组质粒,经转染DH5α细胞筛选阳性菌落后行测序鉴定。将pLentilox3.7U6-TERT重组质粒转染293T细胞,包装产生重组慢病毒Le-TERT并测定其滴度,再用Le-TERT感染大鼠脊髓星形胶质细胞,实时定量-聚合酶链反应检测各细胞株TERT基因表达水平,并利用Western blot和免疫荧光分别检测TERT蛋白的表达。结果与结论:基因测序鉴定证明,pLentilox3.7U6-TERT重组质粒构建成功,并成功包装慢病毒。实时定量PCR、Western blot和免疫荧光检测结果表明,Le-TERT转染星形胶质细胞4 d后,对TERT mRNA的干扰效率可达(63.98±2.6)%,Le-TERT在转然后的星形胶质细胞中呈低表达。结果证实,实验构建的重组表达载体pLentilox3.7U6-TERT能够产生有效滴度的慢病毒,感染大鼠脊髓星形胶质细胞后,该载体可以有效抑制TERT的表达。BACKGROUND：Telomerase reverse transcriptase （TERT） plays an important role in telomerase activation, however there is rare report addressing the construction of the lentivirus targeted its genes to inhibit its expression in the spinal cord astrocytes.OBJECTIVE：To construct recombinant lentivirus vector expressing smal interfering RNA against TERT gene and to evaluate its potential for inhibiting the TERT expression.METHODS：After shRNA-TERT sequence was designed and synthesized, the sequence was amplified by PCR and then connected to plasmid pLentilox3.7U6-hTERT to construct recombinant plasmid. The recombinant plasmid was then transfected to DH5αcel s to screen positive colony, and the sequence was identified. The recombinant plasmid pLentilox3.7U6-TERT was transfected in 293T cel s, generating recombinant lentivirus Le-TERT. The titer of recombinant lentivirus was determined and Le-TERT was transfected into the rat spinal cord astrocytes. The expression of TERT in astrocytes was detected by RT-PCR, western blot and immunofluorescence assay.RESULTS AND CONCLUSION：The gene sequencing analysis confirmed that, recombinant plasmid pLentilox3.7U6-TERT was successful y constructed. The real-time quantitative PCR, western blot analysis and＆amp;nbsp;immunofluorescence assay indicated that, after Le-TERT was transfected in the astrocytes for 4 days, the inhibition rate of TERT mRNA was （63.98±177;2.6）%, and Le-TERT was lowly expressed in the transfected astrocytes. Recombinant expression vector pLentilox3.7U6-TERT can produce the lentivirus at high titer and effectively inhibit TERT expression in the transfected astrocytes.

关 键 词：组织构建 组织工程 组织构建基础实验 RNA干扰 端粒酶 端粒酶反转录酶 质粒载体 慢病毒 星形胶质细胞 短发夹RNA 脊髓损伤 胶质瘢痕 国家自然科学基金 

分 类 号：R394.2[医药卫生—医学遗传学]