小鼠信号转导与转录激活因子3β基因重组腺病毒质粒的构建及其在小鼠乳腺癌4T1细胞中的表达  

Construction of recombinant adenovirus vector for signal transducer and activator of transcriptions 3β gene and its expression in mouse breast cancer 4T1 cells

作　　者：唐浩[1] 党微旗[1] 曹红[1] 王林[1] 陈婷梅[1] 

机构地区：[1]重庆医科大学教育部临床检验诊断学重点实验室,重庆400016

出　　处：《中国生物制品学杂志》2013年第12期1729-1733,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金面上项目(81272544);重庆市科委自然科学基金(cstc2012jjA10011)

摘　　要：目的构建信号转导与转录激活因子3(signal transducer and activator of transcription 3,STAT3)β基因重组腺病毒质粒,并在小鼠乳腺癌4T1细胞中进行表达。方法用HindⅢ和XbaⅠ双酶切质粒pcDNA-Zeo-STAT3β-C4 flag,获得目的基因STAT3β,克隆至穿梭质粒pAdTrack-CMV中,将构建正确的重组穿梭质粒pAdTrack-CMV-STAT3β经PmeⅠ酶切线性化,转化至含腺病毒骨架质粒pAdEasy-1的感受态大肠埃希菌(E.coli)BJ5183中,获得重组腺病毒质粒pAd-STAT3β,转染HEK293A细胞,包装出重组腺病毒,经扩增、CsCl梯度离心纯化后,检测病毒滴度。收集病毒,以50 MOI感染小鼠乳腺癌4T1细胞,RT-PCR及Western blot法检测STAT3β水平。结果重组穿梭质粒经双酶切及测序鉴定,证明构建正确;重组腺病毒质粒经单酶切鉴定,证明构建正确;扩增、纯化后重组腺病毒滴度可达1.1×1013pfu/ml。重组腺病毒Ad-STAT3β感染的小鼠乳腺癌4T1细胞,在转录和蛋白水平上均有STAT3β基因的表达。结论成功构建了重组腺病毒质粒pAd-STAT3β,并在小鼠乳腺癌4T1细胞中表达目的基因STAT3β,为进一步研究乳腺癌的治疗奠定了基础。Objective To construct a recombinant adenovirus vector for signal transducer and activator of transcriptions 3β （STAT3β） gene and express in mouse breast cancer 4T1 cells. Methods Plasmid peDNA-Zeo-STAT3β-C4 flag was digested with Hind Ⅲ and Xba Ⅰ , and the obtained STAT3β gene was cloned to shuttle vector pAdTrack-CMV. The constructed recombinant shuttle plasmid pAdTrack-CMV-STAT3β was linearized with Pac I and transfected to competent E. coli BJ5183 containing adenovirus backbone plasmid pADEasy-1. The obtained recombinant adenovirus plasmid pAd- STAT3βand transfected to HEK293A cells for packaging. The obtained recombinant adenovirus Ad-STAT3[3 was propa- gated and purified by cesium chloride centrifugation, then determined for titer. 4T1 cells were infected with Ad-STAT313 at a MOI of 50, and determined for expression of STAT3β at mRNA and protein levels by RT-PCR and Western blot respectively. Results Restriction analysis and sequencing proved that recombinant shuttle plasmid pAdTrack-CMV- STAT3β was constructed correctly. Recombinant adenovirus vector pAd-STAT3β was constructed correctly as proved by restriction analysis. The titer of recombinant adenovirus Ad-STAT3β reached 1. 1× 10^13 pfu / ml after propagation and purification. The expression of STAT3β at both mRNA and protein levels were observed in 4T1 cells infected with Ad- STAT3β. Conclusion Recombinant adenovirus vector pAd-STAT3β was constructed correctly and expressed in 4T1 cells, which laid a foundation of further study on therapy of breast cancer.

关 键 词：信号转导与转录激活因子3β基因 腺病毒 基因表达 

分 类 号：Q782[生物学—分子生物学]