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目的构建E6AP蛋白结构域突变真核表达载体,并瞬时转染肿瘤细胞,对其生物学功能进行初步检测。方法以本实验室保存带有myc标签的E6AP质粒为模板,采用PCR技术扩增出E6AP(1~494)结构域和E6AP(495~852)结构域(即E6AP的HECT结构域)编码区(CDS)序列,将其插入到p XJ-40-myc载体中,双酶切和测序验证后瞬时转染人胚肾293T细胞,通过Western印迹鉴定其表达及功能;转染人乳腺癌细胞ZR75-1和人胃癌细胞BGC-823,通过CCK-8法测定细胞生长曲线。结果从E6AP蛋白全长CDS中扩增得到约1479、1071 bp的DNA结构域片段,并成功克隆至p XJ-40-myc载体上,且测序与目的序列完全一致;转染人胚肾293T细胞后,目的蛋白在细胞内成功表达,并且E6AP(495~852)结构域可明显抑制P53蛋白水平,而E6AP(1~494)结构域无抑制作用;细胞生长曲线结果显示,转染E6AP(495~852)结构域突变质粒的乳腺癌、胃癌细胞较空载体细胞生长差异具有统计学意义,而E6AP(1~494)对细胞生长无明显变化。结论成功构建了myc-E6AP(1~494)、(495~852)结构域突变真核表达载体,为进一步研究E6AP蛋白在相关肿瘤发生、发展中的作用奠定一定的基础。
Objective To construct eukaryotic expression vector of E6AP protein domain mutation and transiently transfected into tumor cells for the preliminary detection of its biological function. Methods The E6AP plasmids with myc tag in our laboratory were used as templates to amplify the coding region of E6AP (1 ~ 494) and E6AP (495 ~ 852) domains (ie, the HECT domain of E6AP) CDS) was inserted into pXJ-40-myc vector and transiently transfected into human embryonic kidney 293T cells by double enzyme digestion and sequencing. The expression and function of CDS were confirmed by Western blotting. The transfected human breast cancer cells ZR75- 1 and human gastric cancer cell line BGC-823, the cell growth curve was determined by CCK-8 method. Results A DNA fragment of 1479 and 1071 bp was amplified from full-length CDS of E6AP protein and successfully cloned into pXJ-40-myc vector. The sequencing was consistent with the target sequence. The 293T cells transfected with human embryonic kidney The E6AP (495 ~ 852) domain could significantly inhibit the level of P53 protein, while the E6AP (1 ~ 494) domain had no inhibitory effect. The results of cell growth curve showed that E6AP (495 ~ ~ 852) domain mutant plasmids in breast cancer and gastric cancer cells compared with the empty vector was statistically significant, while E6AP (1 ~ 494) had no significant effect on cell growth. Conclusion The myc-E6AP (1-494) and (495-852) domain-mutated eukaryotic expression vectors were successfully constructed, which laid a foundation for further study on the role of E6AP in the development and progression of related tumors.