CEA启动子启动双自杀基因CD/TK重组AdEasy XL腺病毒构建

The construct of replication deficient AdEasy XL adenovirus containing CEA promoter,CD and TK gene

目的构建含有CEA启动子(CEApromoter,Cp)启动的双自杀基因CD与TK的复制缺陷型腺病毒。方法设计带有限制性内切酶酶切序列的PCR引物。在2μlT4DNAligase作用下,Cp插入pAdTrack(pAT)的MCS,形成pAT.Cp;CD插入pAT.Cp的MCS,形成pAT.Cp.C;TK插入pAT.Cp.C的MCS,形成pAT.Cp.C.T。5μgpAT.Cp.C.T经4μlPmeI线化后,转化BJ5183AD1,1μlPacI酶切鉴定。重组AdEasy质粒转化XL10Gold细胞,大量扩增提取纯化,转染5×105AD293细胞,荧光显微镜观察,制备带有Cp.C.T重组腺病毒(recombinantadenoviruswithCp.C.T,RACp.C.T),测定病毒滴度。结果pAT.Cp,pAT.Cp.C,pAT.Cp.C.T双酶切以及PCR均见长约500bp的Cp,1300bp的CD,1100bp的TK。对照质粒DNA转化BJ5183AD1效率为7.36×106cfu/μg。线化pAT.Cp.C.T转化BJ5183AD1,形成重组AdEasy质粒DNA,PacI酶切见典型长约3.0或4.5kb与30.0kbDNA片断。重组质粒转染AD293出现绿色荧光。RACp.C.T滴度为5.67×107pfu/ml。结论RACp.C.T构建正确;AdEasyXL腺病毒载体系统转导Cp.C.T具有高效性,易于观察转染效果。

Objective To construct replication deficient adenovirus containing CD and TK gene promoted by CEA promoter （Cp）. Methods PCR primers of Cp, CD and TK, whose lateral sides were decorated with restriction endonuclease sequences, were designed. T4 DNA ligase （2μl） catalyzed the joint of Cp and pAT to form pAT. Cp, CD and pAD. Cp to form pAT. Cp. C and TK and pAT. Cp. C to form pAT. Cp. C. T. After linerlization with 4μl Pme I, 5μg pAT. Cp. C. T transformed permissive BJ5183-AD-1. The recombinant AdEasy plasmid was amplified with XL 10-Gold and transformed 5 x 105 AD-293 cells. The green fluorescence of AD-293 cells was observed. The titer of recombinant adenovirus with Cp. CD. TK （RA-Cp. CD. TK） was calculated. Results The Cp, CD and TK, whose length was about 500 bp, 1 284 bp and 1131 bp, respectively, were identified with corresponding double endonuclease and PCR. The efficiency of control plasmid DNA transforming BJ5183-AD-1 was 7.36 × 10^6 cfu/μg. The digested pAT. Cp. C. T transformed BJ5183-AD-1, and formed recombinant AdEasy plasmid DNA. The latter was digested by Pac I endonuclease, and the typical DNA segments, whose length was about 3 or 4.5 kb and 30 kb, respectively. AD-293 gave out green fluorescence. Virus titer was 5.67 × 10^7 pfu/ml. Conclusion The RA-Cp. C. T was correctly constructed; AdEasy XL adenoviral vector system was employed to transfer Cp. C. T effectively and easy to be oberserved.