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目的构建CD147-shRNA重组质粒并检测其对肝癌细胞SMMC-7721和HepG2内源性CD147表达的抑制作用。方法根据Genebank中CD147序列设计3条特征性靶序列,合成的核苷酸序列连接到线性化pBS/U6载体,采用酶切法及测序鉴定重组质粒的正确性;将重组质粒转染至人肝癌细胞SMMC-7721和HepG2中,采用Western blot法和免疫荧光法观察该重组质粒对CD147内源性表达的抑制作用。结果酶切鉴定和测序证实成功构建pBS/U6/CD147-shRNA,且对人肝癌细胞SMMC-7721和HepG2 CD147表达有抑制作用,而以pBS/U6/CD147-shRNA3抑制作用最为显著。结论构建pBS/U6/CD147-shRNA成功,并筛选出基因抑制效果最佳的pBS/U6/CD147-shRNA3,为进一步实验打下了基础。
Objective To construct the recombinant plasmid of CD147-shRNA and test its inhibitory effect on the expression of endogenous CD147 in hepatocellular carcinoma cells SMMC-7721 and HepG2. Methods According to the sequence of CD147 in Genebank, three characteristic target sequences were designed. The synthesized nucleotide sequence was ligated into the linearized pBS / U6 vector. The correctness of the recombinant plasmids was confirmed by restriction enzyme digestion and sequencing. The recombinant plasmids were transfected into human hepatocellular carcinoma Cell SMMC-7721 and HepG2, Western blot and immunofluorescence were used to observe the inhibitory effect of the recombinant plasmid on the endogenous expression of CD147. Results Restriction endonuclease digestion and DNA sequencing confirmed that pBS / U6 / CD147-shRNA was successfully constructed and inhibited the expression of SMMC-7721 and HepG2 CD147, while pBS / U6 / CD147-shRNA3 had the most significant inhibitory effect. Conclusion The successful construction of pBS / U6 / CD147-shRNA and the screening of pBS / U6 / CD147-shRNA3 with the best gene inhibition laid the foundation for further experiments.