Construction and verification of RNA interference lentiviral vector targeting eukaryotic elongation factor 2 gene
10.13200/j.cnki.cjb.004288
- VernacularTitle:真核细胞延长因子2基因RNA干扰慢病毒质粒的构建及验证
- Author:
ZHANG Tingting
- Publication Type:Journal Article
- From:
Chinese Journal of Biologicals
2024;37(9):1050-1055
- CountryChina
- Language:Chinese
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Abstract:
Objective To construct RNA interference(RNAi)lentivirus plasmid of eukaryotic elongation factor 2(eEF2)gene and stably transfected breast cancer cell model,so as to provide experimental basis for further study on the role of eEF2in the occurrence and development of breast cancer.Methods According to eEF2 gene sequence and the design principles of short hairpin RNA sequence,three pairs of shRNA sequences were designed and synthesized,which were refolded and inserted into lentivirus vector LV-U6-shRNA-ZSgreen-Puro. Three recombinant plasmids sh1,sh2 and sh3 with different knock-down targets of eEF2 gene were constructed,and empty plasmid vectors were used as negative control group(shNC). Human renal epithelial cells HEK293T were co-transfected with three lentivirus plasmid packaging system for virus packaging and amplification respectively. After identification by digestion and sequencing,the recombinant lentivirus was used to infect breast cancer cells MCF-7 in logarithmic growth period. After 72 h,the green fluorescence intensity was observed under a fluorescence microscope,the mRNA transcription level of eEF2 was determined by fluorescence quantitative PCR(qPCR),and the expression level of eEF2 protein was detected by Western blot.Results The sequence of eEF2shRNA vector was identical to the original sequence. The expression of green fluorescent protein was observed in breast cancer cells MCF-7 infected with recombinant lentivirus. Compared with shNC group,the mRNA transcription levels of eEF2 in sh2 and sh3 group MCF-7 cells with eEF2 knockdown decreased significantly(t = 9. 244 and 5. 938,P = 0. 001and 0. 004,respectively). The expression levels of eEF2 protein in sh1,sh2 and sh3 groups decreased significantly(t =3. 552,9. 614 and 4. 432,P = 0. 024,0. 001 and 0. 011,respectively).Conclusion The lentivirus vector with low expression of eEF2 gene and the stably transfected breast cancer cell model were successfully constructed,which is expected to provide a new target for clinical treatment of breast cancer based on translation-targeted therapy.
