摘要
目的构建新基因CA916798真核表达载体,并观察其对H446增殖的影响。方法以人A549细胞总RNA为模板,用RT-PCR技术扩增获得CA916798开放阅读框序列,连接入pBluescriptⅡSK克隆载体上,将重组质粒转化入大肠杆菌DH5α内并提取质粒,酶切与DNA测序鉴定准确,再用双酶切方法将CA916798开放阅读框序列定向连接到真核表达载体pQE-Trisystem中,构成pQE-Tri-CA916798,将pQE-Tri-CA916798转化入大肠杆菌DH5α内并提取质粒,双酶切鉴定、DNA测序鉴定准确,脂质体转染人小细胞肺癌细胞系H446细胞,选取稳定转染不同质粒的H446细胞,采用MTT绘制细胞增殖曲线,流式细胞仪检测细胞周期和细胞凋亡率。结果扩增出CA916798开放阅读框序列,大小约为350bp,重组质粒的酶切鉴定结果与预期一致,测序结果完全正确。转染了pQE-Tri-CA916798的H446细胞顺铂作用后增殖速度加快,凋亡率下降。结论成功构建了pQE-Tri-CA916798真核表达质粒,并初步证实了其与顺铂诱导的肺癌多药耐药相关。
Objective To observe the effect of CA916798 eukaryotic expression vector transfection on the growth of H446 cells in vitro and explore the role of CA916798 in the MDR of lung cancer. Methods Total RNA was isolated from A549 cells. The ORF of CA916798 was amplified by RT-PCR, then cloned into pBluescript Ⅱ SK vector. After identified by restriction endonucleases digestion and sequencing, it was cloned to the expression vector： pQE-Trisystem vector. The recombinant pQE-Tri-CA916798 was transformed into E. coli DH5α, identified by double digestion of restriction endonucleases and sequencing, then transfected into H446 cells by lipofectamine method. The growth curve was plotted and the sensitivity of cell lines to the drug was detected by MTT test. The effect of CA916798 on the cell cycle was observed by flow cytometry. Results Fragments about 350 bp were amplified. Enzymatic digestion showed that the length was in consistence with the expected. After CDDP treatment, the growth of pQE-Tri-CA916798 transfected cells was faster than that of vector transfected cells, and the cell apoptosis decreased. Conclusion The eukaryotic expression vector was successfully constructed. CA916798 may play an important role in MDR of lung cancer.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2007年第24期2315-2318,共4页
Journal of Third Military Medical University
基金
国家自然科学基金(30470773)~~
