mTERT和mTyr双启动子联合调控HN基因重组腺病毒的构建  

Construction of a recombinant adenovirus with HN gene co-regulated by mTERT and mTyr double promoters

作　　者：龙雨清[1] 李萌[1] 张东超[1] 弓建芳 李小慧[1] 高珂珂 金天明[1] 

机构地区：[1]天津农学院动物科学与动物医学学院,天津300384

出　　处：《中国兽医学报》2016年第5期739-745,共7页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31072109);天津市自然科学基金资助项目(12JCZDJC22100);天津市兽医生物技术优秀科研创新团队基金资助项目(TD12-5019);天津市兽医学重点学科"领军人才培育计划"人选项目

摘　　要：选择新城疫病毒(NDV)的血凝素-神经氨酸酶(HN)基因作为黑色素瘤的治疗基因。将小鼠端粒酶逆转录酶(mTERT)启动子、小鼠黑色素瘤酪氨酸酶(Tyr)特异性启动子核心片段以及HN基因分别插入到腺病毒载体穿梭载体pShuttle-IRES-hrGFP-2的多克隆位点处,将克隆成功的穿梭载体经Pme I线性化后,与腺病毒骨架质粒pAdeasy-1在BJ5183感受态细胞中进行同源重组,成功构建了肿瘤特异性和组织特异性双启动子调控的HN基因的重组腺病毒载体pAd-mTERTp-mTyrp-HN。将重组腺病毒通过脂质体法转染HEK 293细胞,经RT-PCR鉴定及Western-blotting对HN蛋白的表达水平分析后,大量扩增病毒,经CSCl密度梯度超速离心法纯化病毒颗粒,以50%组织培养感染剂量法(TCID50)测定重组腺病毒Ad-HN、Ad-mTERTp-HN、Ad-Tyrp-HN、Ad-mTERTp-TyrpHN和Ad-GFP的最终滴度分别为108.7、109.5、109.25、109.625和109.125,将为下一步利用该重组腺病毒在体内外诱导小鼠黑色素瘤细胞系B16的凋亡及研制动物肿瘤性疾病的基因疫苗提供了理论依据。Melanoma is a kind of common skin malignant tumor,its incidence covers 1%-3% of all malignant tumor.It has a high degree of malignant,and its metastasis always occurs early and widely.It also has strong resistance to conventional treatment,which is a kind of tricky on clinical treatment of malignant tumor.In this research,we chose hemagglutinin-neuraminidase（HN）gene from Newcastle diseases virus（NDV）for the treatment of melanoma.The mouse telomerase reverse transcriptase（mTERT）promoter,the mouse tyrosinase（Tyr）specificity promoter of melanoma and HN gene were inserted into the multiple cloning site（MCS）of adenovirus shuttle vector（pShuttle-IRES-hrGFP-2）,respectively.Linearizing shuttle vector with Pme I,transfected it with pAdeasy-1in BJ5183 cells by homologous recombination,finally obtained recombinant adenovirus vector Ad-mTERTp-Tyrp-HN.The recombinant adenovirus was transfected to HEK 293 cells with Lipofectamine 2000,and obtained a recombinant adenovirus with hemagglutinin-neuraminidase（HN）gene which is co-regulated by mTERT and mTyr promoters named Ad-mTERTpmTyrp-HN.After confirmed by RT-PCR and Western-blotting,collected the virus particles by CSCl density gradient ultracentrifugation and purification,and determined the recombinant adenovirus titers with TCID50 method.The titers of Ad-HN,Ad-mTERTp-HN,Ad-mTyrp-HN,AdmTERTp-Tyrp-HN,Ad-GFP are 108.7,109.5,109.25,109.625 and 109.125 respectively.This research provided both theoretical and experimental basis for in vitro/in vivo melanoma suppression by using recombinant adenovirus,and developed a broad spectrum vaccine for targeting therapy method for animal tumors.

关 键 词：重组腺病毒 HN基因 mTERT启动子 mTyr启动子 

分 类 号：S852.65[农业科学—基础兽医学]