Lck反义RNA重组腺病毒载体的构建及其对肾小管上皮细胞Lck表达的影响

目的:观察淋巴细胞特异性蛋白酪氨酸激酶(Lck)在肾小管上皮细胞中的表达及Lck反义RNA对其表达的影响.方法:采用RT-PCR方法从肾小管上皮细胞中获得Lck目的基因片段,将目的基因片段反向插入pDC 316质粒中,构建Lck反义RNA穿梭质粒(pDC 316-antisenseLck),经PCR、酶切及测序鉴定正确后,将此穿梭质粒与辅助质粒pBHGloxΔE1,3Cre 共转染HEK 293细胞,重组产生Lck基因反义RNA腺病毒载体(Ad-antisenseLck).用此重组腺病毒感染体外培养的肾小管上皮细胞,Western blotting方法观测此重组腺病毒载体对IL-2诱导的Lck蛋白表达的影响.结果:构建出Lck反义RNA重组腺病毒载体,此重组腺病毒载体感染培养的肾小管上皮细胞后可明显抑制IL-2诱导的Lck蛋白表达(P<0.05),抑制率可达47%.结论:成功构建出Lck反义RNA重组腺病毒载体,此载体可在肾小管上皮细胞内发挥生物学作用,显著阻抑肾小管上皮细胞的Lck蛋白表达.

Construction of Lck Gene Antisense RNA Adenoviral Vector and its Effect on Lck Expression in Renal Epithelial Cells

Objective:The objective of this experiment was to construct recombinant adenoviral vector expressing antisense RNA to Lck gene, and investigate its effect on Lck protein expression in renal tubule epithelial cells (TEC).Methods:The Lck cDNA was obtained from TEC of BALB/C mice by RT-PCR and partial Lck cDNA was inserted into pDC 316 plasmid reversely to construct the antisense RNA shuttle plasmid(pDC 316-antisenseLck).The recombinant plasmid was identified through PCR, restriction endonuclease digestion and DNA sequence analysis. This shuttle plasmid and helper plasmid were co-transfected into HEK 293 cells through lipofectin to produce recombinant adenovirus(Ad-antisenseLck), then the recombinant adenovirus was used to infect the TEC. The influence of the adenovirus on Lck protein expression induced by IL-2 stimulations was observed by th way of immunoblotting.Results:The recombinant adenovirus expressing Lck antisense RNA could inhibit the expression of Lck protein in TEC induced by IL-2(P<0.05).Conclusion:A kind of Recombinant adenovirus expressing Lck antisense RNA is constructed successfully and it could inhibit Lck protein expression induced by IL-2 stimulations in TEC significantly.