论文部分内容阅读
目的C35是一种新型的乳腺癌肿瘤标志基因,在乳腺癌细胞中存在普遍性高表达,可作为乳腺癌早期诊断的标志分子。为了进一步揭示C35基因在乳腺癌的发生和发展中对癌细胞的调控和信号转导机制,本研究拟构建C35基因的酵母双杂交诱饵表达载体,并进行自激活活性和诱饵蛋白毒性检测。方法采用RT-PCR自乳腺癌T47D细胞中克隆C35表达序列,合成3种C35基因片段,连接入诱饵表达载体pGBKT7,采用选择性培养皿和β-半乳糖苷酶(β-Galactosidase)分析检测重组质粒的自激活活性和细胞毒性。结果3种诱饵表达载体经鉴定全部克隆成功,其中C35全长基因的表达产物对酵母细胞具有生长抑制作用,两种截短体C35 1-153和C35 154-348不具有自激活活性和细胞毒性,无需3-AT抑制。结论C35全长基因的诱饵表达载体可能具有抑制酵母细胞自身蛋白表达系统的作用,两种截短体重组载体可应用于乳腺癌T47D cDNA文库双杂交筛选。
Objective C35 is a new type of breast cancer tumor marker gene that is highly expressed in breast cancer cells and can be used as a marker for the early diagnosis of breast cancer. In order to further reveal the mechanism of C35 gene regulation and signal transduction in the carcinogenesis and progression of breast cancer, we constructed the yeast two-hybrid bait expression vector of C35 gene and tested its self-activation activity and bait protein toxicity. Methods The C35 expression sequence was cloned from human breast cancer T47D cells by RT-PCR. Three C35 gene fragments were synthesized and inserted into bait expression vector pGBKT7. The recombinant plasmid pGBKT7 was detected by selective culture dish and β-Galactosidase Plasmid self-activation activity and cytotoxicity. Results All the three bait expression vectors were successfully cloned. The expression products of full length C35 gene had a growth inhibitory effect on yeast cells. The two truncated C35 1-153 and C35 154-348 cells had no self-activation activity and cytotoxicity , Without 3-AT inhibition. Conclusion The bait expression vector of C35 full-length gene may inhibit the self-expression system of yeast cells. The two truncated recombinant vectors can be used to screen the breast cancer T47D cDNA library by two-hybrid screening.