| 刘海霞 魏莉 任晓丽 宋晖△ 陈必良△.KLF4siRNA慢病毒载体构建和鉴定[J].,2016,16(19):3601-3604 |
| KLF4siRNA慢病毒载体构建和鉴定 |
| Construction and Identification of siRNA Lentivirus Vector TargetingKLF4 Gene |
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| DOI： |
| 中文关键词: KLF4 慢病毒载体 siRNA 宫颈癌 |
| 英文关键词: KLF4 Lentivirus Vector siRNA Cervical cancer |
| 基金项目:国家自然科学基金项目(81301921)；陕西省自然科学基金项目(2013JM4023 ) |
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| 中文摘要: |
| 目的：构建上皮锌指蛋白4（Kr俟ppel-like factor 4，KLF4）siRNA 慢病毒载体并进行初步鉴定，为研究KLF4 在宫颈细胞癌中
的分子机制奠定基础。方法：利用公用网站中提供的RNA 干扰序列设计原则，设计4 个RNA 干扰靶点序列，合成含干扰序列的
单链DNA oligo，然后退火配对产生双链，再通过其两端所含酶切位点直接连入酶切后的RNAi 慢病毒载体上；将连接产物转入
制备好的细菌感受态细胞，PCR鉴定阳性重组子后，送测序验证，测序结果经比对确认正确的克隆，制备编码慢病毒颗粒的重组
病毒质粒及其两种辅助包装原件载体质粒，共转染293T细胞，收集富含慢病毒颗粒上清液，对其浓缩后得到高滴度的慢病毒浓
缩液，在293T 细胞中测定并标定病毒滴度。收集上清液感染宫颈癌HeLa 细胞，通过qRT-PCR 及Western Blot 鉴定KLF4 siRNA
慢病毒干扰效果。结果：成功构建KLF4 siRNA 慢病毒载体。KLF4 siRNA 慢病毒感染HeLa细胞后，qRT-PCR 及Western Blot 测
定结果显示，KLF4 表达明显降低。结论：KLF4 siRNA 慢病毒载体构建及包装成功，可有效抑制KLF4 表达，为研究KLF4 生物学
功能奠定基础。 |
| 英文摘要: |
| Objective:To construct and identify the Lentivirus vector interfering human KLF4 gene in order to further research the
function of KLF4 in HeLa cell.Methods:Small interference RNA (siRNA) targetingKLF4 gene was designed and DNA template was
synthesized and subcloned into the Lentivirus expression plasmid. The recombinant plasmid KLF4 siRNA was confirmed by PCR and
gene sequencing. The KLF4 siRNA and Lentiviral packaging plasmid was transfected into 293T cells by liposome. After collecting and
concentrating supernatant culture medium to infect HeLa cells. The infected Hela cells with KLF4 siRNA were identified by qRT-PCR
and Western blot.Results:The recombinant lentivirus expression plasmidKLF4 siRNA were successfully constructed. The recombinant
lentivirus vector KLF4 siRNA could infect HeLa cells successfully. The results of qRT-PCR and Western blot showed that the expression
of KLF4 was obviously inhibited in the HeLa cells infected by KLF4 siRNA lentivirus.Conclusion:The recombinant lentivirus vector
KLF4 siRNA was successfully constructed which could inhibit the expression of KLF4. |
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