Ad-HBx的构建及在肾小球足细胞系的表达  

Construction of Recombinant Adenovirus Containing HBx Gene and Its Expression in Podocytes

作　　者：张瑜[1] 周建华[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院儿科,武汉430030

出　　处：《华中科技大学学报（医学版）》2009年第2期213-216,共4页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目(No.30772360)

摘　　要：目的构建携带HBx基因的非复制型重组腺病毒载体,并感染小鼠肾小球足细胞株MPC5,使目的基因HBx在该细胞株有效表达。方法由质粒PCI-neo-HBx中扩增目的基因HBx,插入至pShuttle-CMV(-)穿梭质粒中,而后再与pAdxsi质粒酶切连接为重组腺病毒质粒pAd-HBx,经酶切及测序鉴定正确后,以PacⅠ酶切线性化转染293细胞进行包装扩增得到Ad-HBx。以Ad-HBx感染MPC5细胞,Western blot检测感染后目的蛋白HBx的表达。结果重组腺病毒载体经限制性内切酶酶切、电泳和基因测序鉴定,证实Ad-HBx构建成功;当MOI=50时,MPC5细胞即可100%被重组腺病毒感染,且持续至感染后72 h仍有较强水平的表达;感染Ad-HBx的MPC5细胞可稳定表达HBx蛋白。结论成功构建了Ad-HBx,并能在MPC5细胞中稳定表达,为进一步研究HBx基因在乙型肝炎病毒相关性肾炎发病中的作用奠定了基础。Objective To construct a recombinant adenoviral vector containing HBV X（HBx）gene for specific expression of X gene in transfected mouse podocytes（MPC5 cell line）. Methods HBx cDNA was obtained from the plasmid PCI-neo-HBx by enzyme digestion,and inserted into shuttle plasmid pShuttle-CMV（-） to generate a recombinant plasmid pShuttle-HBx. Then the HBx gene was transferred from pShuttle-HBx to Adxsi viral DNA to form a recombinant adenoviral plasmid pAd-HBx by means of an in vitro ligation reaction. Finally,after identification, the pAd-HBx was packaged into infectious adenoviral particles Ad-HBx by transfecting human embryonic kidney（HEK） 293 cells. Differentiated podocytes were transfected with the adenovirus,and then the expression of HBx protein was detected by Western blot. Results The recombinant adenovirus Ad-HBx was generated successfully by an in vitro ligation reaction, which was confirmed by restriction enzyme digestion and DNA sequencing. The expression of HBx protein in the differentiated podocytes transfected with Ad-HBx was detected by Western blot. Conclusion The successful construction of the recombinant adenovirus containing HBx gene and the effective expression of HBx protein in mouse podocyte cell line have laid a foundation for further study on the involvement of HBx in the podocytopathy in HBV-GN.

关 键 词：HBX基因 腺病毒载体 足细胞 

分 类 号：R692.3[医药卫生—泌尿科学]