CAJ | 학술논문

目的:利用AdEasy~(TM) system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Not I和Hind Ⅲ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pAdEasy~(TM)DNA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Western-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α-SMA 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.
목적:이용AdEasy~(TM) system구건휴대대서BMP-7기인적중조선병독,병감정기재원대신소관상피세포중적표체.방법:장BMP-7전장서렬분위46개편단분단합성,재채용PCR방법장합성적46개과핵감산편단병접성BMP-7기인,병극륭도측서재체중측서감정.경Not I화Hind Ⅲ쌍매절후접입pShuttle-CMV천사재체,구건중조선병독적천사질립pShuttle-BMP-7.EcoRI매절중조pShuttle-BMP-7,여pAdEasy~(TM)DNA전천공공전화BJ5183감수태균, 추제중조AdBMP-7질립,PacI매절선성화중조질립AdBMP-7후,전염Ad293세포진행병독포장화확증,선병독형광검측시제합측정기적도.용AdBMP-7감염대서신소관상피세포,이ELISA법화Western-blot법검측BMP(-7)재대서신소관상피세포중적표체.RT-PCR방법검측α-SMA mRNA적표체수평,이감정중조선병독AdBMP-7적생물학활성.결과:구건료BMP-7기인적중조선병독재체,병제비출고적도중조선병독보존액.해중조선병독재체외능유효전염대서신소관상피세포병고표체BMP-7단백,소표체적BMP-7단백능유효역전유TGF-β1유도적α-SMA표체증고.결론:성공지구건료휴대대서BMP-7기인적중조선병독,병초보증실기구유일정적항신섬유화공능.
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.