大鼠BMP-7重组腺病毒构建及在肾小管上皮细胞中表达

Construction of recombinant adenovirus containing BMP-7 gene and its expression in proximal tubule epithelial cells

目的:利用AdEasyTMsystem构建携带大鼠BMP-7基因的重组腺病毒,并鉴定其在原代肾小管上皮细胞中的表达。方法:将BMP-7全长序列分为46个片段分段合成,再采用PCR方法将合成的46个寡核苷酸片段拼接成BMP-7基因,并克隆到测序载体中测序鉴定。经NotI和HindⅢ双酶切后接入pShuttle-CMV穿梭载体,构建重组腺病毒的穿梭质粒pShuttle-BMP-7。EcoRI酶切重组pShuttle-BMP-7,与pAdEasyTMDNA电穿孔共转化BJ5183感受态菌,抽提重组AdBMP-7质粒,PacI酶切线性化重组质粒AdBMP-7后,转染Ad293细胞进行病毒包装和扩增,腺病毒荧光检测试剂盒测定其滴度。用AdBMP-7感染大鼠肾小管上皮细胞,以ELISA法和W estern-blot法检测BMP-7在大鼠肾小管上皮细胞中的表达。RT-PCR方法检测-αSMA mRNA的表达水平,以鉴定重组腺病毒AdBMP-7的生物学活性。结果:构建了BMP-7基因的重组腺病毒载体,并制备出高滴度重组腺病毒保存液。该重组腺病毒在体外能有效转染大鼠肾小管上皮细胞并高表达BMP-7蛋白,所表达的BMP-7蛋白能有效逆转由TGF-β1诱导的α-SMA表达增高。结论:成功地构建了携带大鼠BMP-7基因的重组腺病毒,并初步证实其具有一定的抗肾纤维化功能。

Objective： To construct a recombinant adenovirus vector containing bone morphogenic protein-7（BMP-7） gene and to identify its biological activities in proximal tubule epithelial cells（PTECs）.Methods： Forty-six fragments of BMP-7 gene were obtained by PCR method and then were ligated to the full-length gene.The full length sequence of BMP-7 was subcloned into pBluescriptⅡ（＋） vectors,and confirmed by sequencing.Double digested with Not I and HindⅢ,BMP-7 gene was inserted into pShuttle-CMV.EcoRⅠ pre-linearized pShuttle plasmid was transformed into competence bacterium BJ5183 to obtain the recombinant adenovirus-BMP-7 by efficient homologous recombination.Then the AdBMP-7 was obtained by packaging PacⅠ linearized in 293 cells.Adenoviral titer was determined by adenovirus fluorescent detection kit.The protein expression of BMP-7 in PTECs was respectively detected by ELISA and Western blot.RT-PCR method was used for analyzing the α-smooth muscle action（α-SMA） expression in PTECs,which was treated consecutively with TGF-β and AdBMP-7.Results： The recombinant plasmid AdBMP-7 was successfully generated,which increased BMP-7 protein expression levels in PTECs,and down-regulated TGF-β-induced α-SMA expression.Conclusion： The bioactive recombinant adenovirus AdBMP-7 has been successfully constructed,which may be effective in inhibition of chronic renal fibrosis.