人IL-2复制缺陷型重组腺病毒的构建和鉴定  

Construction and identification of human interleukin-2 replication-deficient recombinant adenoviruses

作　　者：孙运祥[1] 任丽君[1] 郭玉嵩[1] 田长富[1] 

机构地区：[1]哈尔滨医科大学肿瘤防治研究所,哈尔滨150040

出　　处：《实用肿瘤学杂志》2005年第4期260-263,共4页Practical Oncology Journal

基　　金：黑龙江省科技攻关基金(GB02C148-02;SY02-06);哈尔滨市科学基金(2002AFXYJ058)资助

摘　　要：目的构建含hIL-2的复制缺陷型重组腺病毒。方法将hIL-2DNA片段与载体pDNR-Dual-CMV融合,转化大肠杆菌DH5α。得到重组质粒(pDNR-Dual-hIL-2),经鉴定正确,将pDNR-Dual-hIL-2质粒与pLP-Adeno-x-CMV质粒重组得到重组载体pLP-Adeno-hIL-2,再次转化大肠杆菌DH5α,挑取正确克隆,大量扩增,提取质粒。将此质粒经PacⅠ酶切后与LipofectamineTM2000转染HEK293细胞,产生含人IL-2的复制缺陷重组腺病毒(AdhIL-2)。结果pLP-Adeno-hIL-2重组载体经PCR、酶切鉴定结果与预期相符,所得重组病毒滴度达到5×107PFUml。结论成功构建了hIL-2的复制缺陷型重组腺病毒。Objective To construct the hIL - 2 replication - deficient recombinant adenoviruses. Methods Recombine hIL - 2 DNA with pDNR - Dual - CMV vector and then transformed into E. coil DH5α, attain the reeombined vector- pDNR - Dual - hIL- 2. It was identified that vector had been constructed suceessfully. Recombined the plasmid pDNR - Dual - hIL - 2 and pLP - Adeno - x - CMV attained the recombinant adenovirus vector - pLP - Adeno - hIL - 2, transferred the plasmid into E. coli DH5α. Extract the reeombined plasmid, then digested by Pac I , the recombined adenoviruses vector was transfected into HEK 293 cells together with Lipafectamine^TM 2000, then harvest the hIL - 2 replication - deficient recombinant adenoviruses. Results The analysis of PCR and restriction enzyme digestion of recombinant adenovirus vector were completely identical to the results that were anticipated. The recombinant adenovirus was obtained with the titers of 5 × 10^7 PFU/ml. Conclusion Human interleukin - 2 replication - deficient recombinant adenoviruses were construeted.

关 键 词：白细胞介素2 腺病毒载体 同源重组 复制缺陷重组腺病毒 缺陷型重组腺病毒 人IL-2 鉴定结果 HEK293细胞 HIL-2 重组质粒 

分 类 号：R73-3[医药卫生—肿瘤]