小鼠趋化因子受体7特异性shRNA腺病毒表达载体的构建和鉴定

CONSTRUCTION AND IDENDIFICATION OF MOUSE CC-CHEMOKINE RECEPTOR 7-SPECIFIC shRNA ADENOVIRAL EXPRESSION VECTOR

目的构建小鼠CC趋化因子受体7(CCR7)基因特异性短发卡RNA(shRNA)真核表达载体,研究其对CCR7分子表达的影响。方法根据小鼠CCR7 mRNA序列,设计三个shRNA序列,并插入表达载体p HBAd-U6-GFP中,经基因测序鉴定。然后用获得的重组CCR7 shRNA表达载体转染人胚肾细胞HEK293,进行病毒包装、出毒和扩增,测定病毒滴度TCID50,并用重组腺病毒感染小鼠MEF细胞,观察细胞绿色荧光强度,Western Blotting检测CCR7蛋白的表达。结果基因测序证明:获得的三个shRNA载体序列与原设计序列完全相符,扩增后的病毒上清滴度分别为:1.0×1010、1.6×1010和1.25×1010PFU/m L。Ad CCR7 shRNA1腺病毒可明显下调小鼠MEF细胞CCR7蛋白表达。结论成功构建了小鼠CCR7特异性shRNA腺病毒表达载体,并获得了高滴度病毒上清,为以后研究肿瘤淋巴转移的分子机制和信号通路打下了坚实的基础。

Objective To construct eukaryotic expression vector encoding short hairpin RNA （shRNA） specific for the mouse cc - chemokine receptor 7 （CCR7） gene and research on its effect of the expression in CCR7 mol- ecules. Method Three shRNA sequences, shRNA1, shRNA2 and shRNA3 based on the sequence of mouse CCR7 mRNA were designed and inserted into shRNA expression vector pHBAd - U6 - GFP by gene sequencing. Then the recombinant CCR7 shRNA expression vectors, CCR7 - shRNA1/2/3, and backbone vector pHBAd - BHG transfect- ed human embryonic kidney cells, HEK 293, to process virus package, release and amplification and further examine and confirm the viral titer TCIDSO. At the same time, apply recombinant adenoviral to contaminatemouse MEF cell, observe cell＇s green fluorescence intensity and detect the expression of CCR7 protein by Western Blotting. Result Proved by gene sequencing： The three shRNA vectors completely correspond to previous designed sequences and the amplified viral titers were respectively 1.0 × 10^10 PFU/mL, 1.6 ×10^10 PFU/mL and 1.25 × 10^10 PFU/mL. CCR7 expression of mouse MEF cell protein was significantly down - regulated by Ad CCR7 shRNA1 adenoviral vector. Conclusion The shRNA adenoviral expression vector specific for mouse CCR7 was successfully constructed and high - titer viral supernatant obtained, which lays a solid foundation for further investigation of the molecular mechanism of tumor lymph metastasis and signal pathways.