目的：制备 shRNA 干扰人长链非编码 RNA Linc00461的慢病毒载体（shRNA-Linc00461），为进一步深入研究长链非编码 RNA 的功能奠定基础。方法利用在线干扰设计网页进行干扰引物设计，根据小干扰 RNA 的设计原则，选取三对针对Linc00461特异性干扰引物送公司合成。合成的片段连接到慢病毒载体质粒 pSIH1-H1，形成 pSIH1-H1-shRNA-Linc00461质粒。用 PCR 及测序对其进行鉴定，之后与慢病毒包装质粒混匀，由脂质体转染至 HEK293T 细胞进行包装，产生慢病毒 shR-NA-Linc00461，收集上清液使其感染乳腺癌 MDA-MB-231细胞，通过 RT-PCR 鉴定重组慢病毒干扰效果。并选取干扰效果最好的 Linc00461感染 MDA-MB-231细胞，运用 MTT 和流式细胞术检测细胞增殖和周期的改变。结果 SIH1-H1-shRNA Linc00461质粒经 PCR 验证和测序后，证实其构建成功。shRNA-Linc00461在 HEK293T 细胞中重组成慢病毒，并成功感染MDA-MB-231，感染效率约为70％。shRNA-Linc00461感染乳腺癌 MDA-MB-231细胞后 Linc00461的表达下调65．32％；感染shRNA-Linc00461第3天 MTT 结果显示：实验组吸光度值（0．232±0．032）明显低于实验对照组（0．398±0．037）。流式结果提示实验组 G2M期细胞（47．55±5．063）％高于实验对照组（8．876±3．565）％。结论成功构建了 shRNA-Linc00461慢病毒载体，并能有效抑制 Linc00461表达，为深入研究人 Linc00461基因功能奠定了基础。%Objective To construct the recombinant lentivirus vector interfering human Linc00461 gene for its further research.Meth-ods Short hairpin RNA(shRNA)targeting Linc00461 gene was designed and DNA template was synthesized and subcloned into the Lentivirus expression plasmid pSIH1-H1.The recombinant plasmid pSIH1-H1-shRNA-Linc00461 was confirmed by PCR and gene se-quencing.Then pSIH1-H1-Linc00461 and Lentiviral packaging plasmid was transfected into HEK293 cells for packaging by liposome. And supernatant culture medium was collected to infect MDA-MB-231 breast cancer cells.shRNA-Linc00461 was identified by RT-PCR.Breast cancer cells MDA-MB-231 were transfected with shRNA-Linc00461 and the cells proliferation and change cell cycle were detected by MTT and FCM.Results The recombinant Lentivirus expression plasmid pSIH1-H1-shRNA-Linc00461 was successfully constructed.The recombinant Lentivirus vector shRNA-Linc00461 could be packaged in HEK293T and infected MDA-MB-231 success-fully.The expression of Linc00461 gene was reduced by 65.32% when the breast cancer MDA-MB-231 cells were infected by shRNA-Linc00461.MTT assay showed that cell proliferation capacity decreased from (0.5780 ±0.047)to(0.432 ±0.042)and blocked cell cycle in G2MPhase was(47.55 ±5.063)%,higher than(8.876 ±3.565)% in control group.Conclusions The recombinant Lenti-virus vector interfering Linc00461 gene was successfully constructed and inhibited the expression of Linc00461,which laid foundation for further research of Linc00461.
展开▼
