摘要
目的构建抑制大鼠ERp29基因表达的重组质粒,并在IEC-6细胞中发挥作用。方法根据大鼠ERp29的基因序列,设计合成含有发夹结构的寡核苷酸片段,经退火形成双链后克隆至pAVU6+27载体质粒中,对阳性重组子进行酶切和测序鉴定后转染正常IEC-6细胞,通过PCR方法对siERp29干扰片段进行鉴定,并采用W estern b lot检测ERp29蛋白水平的表达变化。结果重组质粒经双酶切后,有目的条带出现,提示ERp29干扰片段已克隆至pAVU6+27载体质粒中,DNA测序结果显示插入序列与预先设计完全一致。重组质粒转染IEC-6细胞后经G418筛选,成功获得阳性克隆,W estern b lot结果显示ERp29的蛋白水平表达显著下降。结论成功构建ERp29基因RNA干扰表达质粒,并在IEC-6细胞中发挥抑制作用,这为深入研究该基因在电离辐射中的作用奠定了基础。
Objective To construct pAVU6-siERp29 expression plasmid and observe its function in IEC-6 cells. Methods Rat siERp29 was subcloned into pAVU6 ＋27, DH10B E. coli. was transformed with the recombinant plasmid, and the ampicillin-resistant clones were identified by double digestion with Hindlll and BamHI and DNA sequencing. IEC-6 cells were transfected with the identified pAVU6-siERp29 and pAVU6 ＋27 by Lipofectamine2000. Results The DNA fragment obtained by double digestion and DNA sequencing showed that siERp29 was inserted into the pAVU6 ＋27 in correct orientation and reading frame. G418-resistant colonies of IEC- 6 ceils were obtained and confirmed as positive by PCR. The expression of ERp29 protein decreased by Western blotting. Conclusion pAVU6-siERp29 expression plasmid was constructed and took effect in IEC-6 cells, which help to further study the biochemical and physiological role in radiation response of this protein.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2006年第11期1135-1137,共3页
Journal of Third Military Medical University
基金
国家自然科学基金资助项目(30230360
30400121)~~
