携带p53AIP1基因的重组复制缺陷型腺病毒载体的构建、鉴定及其在人宫颈癌细胞系HeLa中的表达  

Construction and identification of a recombinant replication-defective adenovirus vector encoding p53AIP1 and its expression in human HeLa cells

作　　者：林晓亮[1,2] 姜云波[2] 赵倩 殷小涛[1,2] 田仁礼[1,2] 李云奇[1,2] 徐元基[2] 阎瑾琦[2] 张巍[2] 高江平[1] 于继云[2] 

机构地区：[1]解放军总医院泌尿外科,北京100853 [2]军事医学科学院基础医学研究所,北京100850 [3]河北省石家庄市新华区计划生育技术服务站,河北石家庄050000

出　　处：《细胞与分子免疫学杂志》2013年第10期1082-1086,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81201612);国家高技术研究发展计划(863)(2007AA02Z451)

摘　　要：目的构建携带外源性p53调节的凋亡诱导蛋白1(p53AIP1)基因重组复制缺陷型腺病毒载体,观察其在HeLa细胞中的表达。方法设计针对p53AIP1基因序列并插入特定酶切位点的特异引物,通过PCR扩增p53AIP1基因序列,利用DNA重组技术将p53AIP1基因定向插入到腺病毒穿梭载体pDC316,构建穿梭质粒pDC316-p53AIP1;在LipofectamineTM2000介导下穿梭载体pDC316-p53AIP1和腺病毒骨架载体pBHGloxΔE1,3Cre共转染进HEK293细胞;通过Cre-loxP重组酶系统,使Shuttle质粒和辅助大质粒发生定点重组从而包装产生携带目的基因p53AIP1的复制缺陷型重组腺病毒(Ad-p53AIP1)。将Ad-p53AIP1和Ad-null按MOI=100感染HeLa细胞,应用Western blot法检测蛋白水平的表达。结果基因片段成功连入pDC316载体上,对所构建新载体分别行PCR扩增、酶切、测序鉴定,与预计一致。Western blot法检测证实,Ad-p53AIP1感染HeLa细胞后蛋白有表达。结论成功构建携带p53AIP1基因的重组复制缺陷型腺病毒载体,并且有较强的感染能力。Objective To construct a recombinant replication-defective adenovirus vector carrying p53AIP1 （p53-regulated apoptosis-inducing protein 1 ） gene and observe its expression in human HeLa cells. Methods Specific primers were designed according to p53AIP1 gene sequence and inserted specific enzyme cutting sites. P53AIPt gene was amplified by PCR and cloned into the adenovirus shuttle plasmid pDC316 to construct the recombinant vector pDC316-p53AIP1, which was co-transfected with helper plasmid pBHGIoxAE], 3Cre into HEK293 cells by LipofectamineTM 2000. The recombinant replication-defective adenovirus （Ad-p53AIP1） was generated by means of homologous recombination of the two plasmids in HEK293 cells with the Cre-loxP recombinase system and harvested after 12 days. Ad-p53AIP1 and Ad-null were respectively transfected into HeLa cells at MOI = 100. Then the expression of p53AIP1 gene was detected by Western blotting. Results pDC316-p53AIP1 was constructed successfully. The new constructed vector was confirmed by PCR analysis, double enzyme digestion and DNA sequencing. The results were in conformity with the expected. Western blotting demonstrated that the target p53AIP1 proteins were effectively expressed in the transfected HeLa cells. Conclusion The recombinant adenovirus vector of Ad-p53AIP1 was successfully established, and it was proved to have a strong ability of infectivity.

关 键 词：p53调节的凋亡诱导蛋白1 腺病毒载体 HELA细胞 基因治疗 

分 类 号：R392.12[医药卫生—免疫学] Q786[医药卫生—基础医学]