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目的构建带有Flag标签的生长抑制肽(34p)的真核表达载体,并验证其表达与功能。方法抽提人肝癌细胞系HepG2的基因组DNA为模板进行PCR扩增目的片段,通过酶切连接,将DNA片段连接到p3XFLAGCMVTM-9表达载体上,进行测序验证;随后进行瞬时转染,通过免疫荧光实验验证该载体的表达情况,用流式细胞术实验证实其功能。结果测序结果显示载体构建成功,序列正确;免疫荧光结果证实该载体可以正常表达目的肽段;流式细胞术结果过表达34p组相较于转染空载组,细胞凋亡明显增加。结论成功构建了34p的真核表达载体,并验证其促凋亡的生物学功能,为后续的机制研究提供了研究手段和工具。
Objective To construct a Flag-tagged growth inhibitory peptide (34p) eukaryotic expression vector and verify its expression and function. Methods Genomic DNA extracted from HepG2 cell line was used as a template to amplify the target DNA fragment. The DNA fragment was ligated into p3XFLAGCMVTM-9 expression vector and verified by sequencing. Then, transient transfection was performed, The expression of the vector was verified by experiments, and its function was verified by flow cytometry. Results The sequencing results showed that the vector was successfully constructed and the sequence was correct. The results of immunofluorescence showed that the vector could express the target peptide normally. The results of flow cytometry showed that the apoptosis of the 34p group was significantly increased compared with the 34p group. Conclusion The eukaryotic expression vector of 34p was successfully constructed and its biological functions of proapoptosis were verified, which provided the research tools and tools for the subsequent mechanism research.