人磷酸二脂酶基因反义RNA重组腺病毒载体的构建

Construction of an Antisense RNA Recombinant Adenovirus Vector of the Human PDE5A1 Gene Promoter

目的探讨人磷酸二脂酶5(PDE5)基因反义RNA重组腺病毒载体的构建方法。方法根据基因库确定具有完整PDE5A1启动子活性的cDNA基因序列,并设计反义链,两'端加内切酶修饰;通过转载体克隆方法构建转入载体(pENTR)并测序;借助Gateway腺病毒表达载体系统进行重组反应构建重组腺病毒表达载体pAd/CMV/V5/antisense PDE5A1;PacⅠ酶切后转染293A细胞系进行包装、扩增;病毒质粒酶切后电泳鉴定及PCR检测;CsCl梯度离心法纯化病毒,病毒空斑实验进行滴度测定。结果pENTR载体测序证实目的基因序列及插入方向正确;pAd/CMV/V5/antisense PDE5A1酶切后电泳鉴定,可见145bp的片段;PCR检测证明实验设计的目的反义基因成功插入腺病毒载体中;重组腺病毒滴度达108～1010/μl。结论借助Gateway腺病毒表达载体系统可成功地将人工合成的反义基因装入到腺病毒载体中;纯化病毒液量和滴度符合体内基因转染实验的要求。

Objective: To construct an antisense RNA recombinant adenovirus vector of the human PDE5A_~1 promoter gene. Methods: A cDNA fragment containing the human PDE5A_~1 promoter and the human PDE5A1-specific exon was determined according to the gene bank. The antisense RNA fragment was synthesized artificially and subcloned into the pENTR. Then, the sequence of pENTR fragement was detected, and the recombinant adenovirus vector pAd/CMV/V5/antisense-PDE5A_~1 was constructed by LR reaction with the Gateway expression system. The identified recombinant adenovirus plasmid was digested with Pac Ⅰand transformed into 293A cells to package recombinant adenovirus particles. The recombinant adenovirus particles were tested with PCR technique and purified after acquisition by CsCl density gradient ultracentrifugation. Results: The sequencing result showed a 145 bp fragment in pENTR, which was proved to be the domain of the antisense RNA fragment.PCR confirmed that the antisense RNA fragment was cloned into the recombinant adenovirus vector pAd/CMV/V5/antisense-PDE5A_~1 successfully and could infect 293A cells.The titer of virus stocks was up to 10~8 ～10~10 /μl after purification. Conclusion: With the Gateway expression system, the culturing and reproduction of 293A cells can reproduce recombinant adenovirus pAd/CMV/V5-DEST successfully, and the recombinant adenovirus vector can meet the need of in vivo gene transfection in laboratory studies