摘要
目的构建由具有肿瘤特异性启动作用的端粒酶逆转录酶启动子(mTERT-promotor)驱动m4-1BBL(CD137Ligand)基因的腺病毒载体。方法用PCR和RT-PCR的方法,分别从C57BL/6小鼠组织中克隆mTERT启动子和共刺激分子m4-1BBL基因,通过T载体和转移载体将目的基因亚克隆到腺病毒载体上,293A细胞包装成病毒颗粒rAD-mTERT-m4-1BBL,流式细胞仪检测m4-1BBL基因在Hepa1-6及L929细胞上的表达。结果通过PCR检测、直接测序证实目的基因克隆成功并亚克隆到表达载体上,细胞免疫化法证实病毒包装成功,流式细胞仪检测发现重组腺病毒载体rAD-mTERT-m4-1BBL能特异性地在肿瘤细胞Hepa1-6上高表达m4-1BBL。结论由mTERT-promotor驱动m4-1BBL基因的具有肿瘤特异性的腺病毒载体构建成功,为下一步进行体内外抗肿瘤实验打下了基础。
Objective To construct an adeno virus vector with an inserted aiming gene m4-1BBL （CD137 ligand） drived by telomerase reverse transcriptase promotor （mTERT promotor）. Method Clone aiming gene mTERT promotor and m4-1BBL were obtained by PCR and RT-PCR from C57BL/6 mouse＇s tissue respectively, and the two genes were cloned into an adeno virus expression vector. Results The aiming genes were verificated that they had been cloned and connected into expression vector successively by PCR and direct sequencing, immunoeytochemistry verifieated that the virus was packaged successively and FCM conformed that m4-1BBL was specifically expressed on Hepa 1-6 cells. Conclusion The tumor specific adeno virus vector which has an aiming gene m4-1BBL drived by mTERT promotor is constructed and the foundation of the descend experiment is found.
出处
《苏州大学学报（医学版）》
CAS
北大核心
2009年第3期463-466,F0003,共5页
Suzhou University Journal of Medical Science
基金
江苏省自然科学基金资助项目(BK2005037)
