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目的:定点突变合成p53基因突变子,构建绿色荧光蛋白表达载体,进而观察其在HepG2细胞中与内源性热休克蛋白70的共定位关系。方法:以野生型p53为模板,采用PCR体外定点突变技术通过重叠延伸法2次PCR扩增得到目的基因片段,进一步将其克隆到绿色荧光蛋白载体pEGFP-C3中。将构建好的载体转染到HepG2细胞中,利用免疫荧光染色法检测内源性热休克蛋白70的表达,利用荧光显微镜观察hsp70与p53的共定位关系。结果:测序结果显示片段插入正确,在预期位点发生了点突变,249位氨基酸由AGG突变为AGC,273位氨基酸由CGT突变为CAT。载体成功构建。转染后观察到热休克蛋白70与273Hp53共同定位于肝癌细胞系HepG2的胞质,而wtp53与249M p53均定位于细胞核。结论:热休克蛋白70与不同点突变的p53在肝癌细胞系HepG2中的共定位关系与之前我们在肝癌组织中发现的hsp70与p53共定位关系不同,这些提示在hsp70与p53的共定位关系和相互作用上存在某种尚未阐明的机制。
OBJECTIVE: To mutate the mutant of p53 gene by site-directed mutagenesis to construct a green fluorescent protein expression vector and further to observe its colocalization with endogenous heat shock protein 70 in HepG2 cells. Methods: Wild-type p53 was used as a template to amplify the target gene by PCR in situ. The target gene fragment was amplified by overlap extension method and further cloned into the green fluorescent protein vector pEGFP-C3. The constructed vector was transfected into HepG2 cells, the expression of endogenous heat shock protein 70 was detected by immunofluorescence staining, and the co-localization of hsp70 and p53 was observed by fluorescence microscopy. Results: Sequencing results showed that the fragment was correctly inserted and a point mutation occurred at the expected site. The amino acid at position 249 changed from AGG to AGC and the amino acid at position 273 changed from CGT to CAT. Vector was successfully constructed. After transfection, it was observed that HSP70 and 273Hp53 co-localized in the cytoplasm of hepatoma cell line HepG2, while both wtp53 and 249M p53 were located in the nucleus. CONCLUSION: The co-localization of heat shock protein 70 and p53 with different mutations in HepG2 cell line is different from the colocalization of hsp70 and p53 previously found in hepatocellular carcinoma. These suggest that colocalization of hsp70 with p53 and There is some mechanism of interaction that has not yet been clarified.