摘要
目的:构建大鼠转化生长因子-β1(TGF-β1)基因短发卡RNA干扰慢病毒载体,并通过感染大鼠原代逼尿肌细胞进行鉴定。方法:根据大鼠TGF-β1基因设计合成3对shRNA序列,将其分别连接至带荧光标签和抗性基因的慢病毒载体pHBLV上,通过转化至DH5α感受态细胞,提取阳性克隆后用DNA测序进行鉴定,选择序列正确的克隆大量扩增并提取重组的慢病毒质粒。将重组慢病毒质粒和慢病毒包装的辅助质粒转染至293T细胞进行慢病毒包被,抗性筛选后通过荧光法对病毒进行滴度测定。用慢病毒感染大鼠原代逼尿肌细胞,通过qRT-PCR和Western blot检测TGF-β1 mRNA和蛋白的表达水平。结果:设计的3种大鼠TGF-β1 shRNA干扰慢病毒载体经测序鉴定构建成功。使用该病毒感染大鼠原代逼尿肌细胞,qRT-PCR和Western blot结果显示,与空白对照组和阴性对照组相比,3种TGF-β1 shRNA感染组的细胞TGF-β1在mRNA和蛋白水平均有不同程度的下调,其中TGF-β1 shRNA 2的蛋白敲低效率最好(57%)。结论:本研究成功构建了大鼠TGF-β1 shRNA干扰慢病毒载体,并在大鼠原代逼尿肌细胞中进行了鉴定,为进一步的基因治疗研究提供了可能性的基础。
Objective:To construct the recombinant lentiviral vector for rat TGF-β1 shRNA interference and to detect the expression of TGF-β1 at mRNA and protein level when the packaged lentivirus infected the rat primary detrusor cells.Methods:3 pairs of rat TGF-β1 shRNA were designed and synthetized,then ligated to the lentivirus vector(pHBLV)with fluorescent tags and resistance gene.These recombinant lentivirus plasmids were transformed into the competent cells E.coli DH5α.The positive clones were verified by DNA sequencing.Then the right clones were largely amplified and extracted.The recombinant lentivirus plasmids and the lentivirus packaging plasmids were co-transfected into 293T cells for lentivirus production.Following the resistance screening,the lentivirus titer was determined by the fluorescence method.Then infected the primary detrusor cells were infected with the above lentivirus to measure the expression of TGF-β1 at mRNA and protein level by qRT-PCR and Western blot.Results:Three kinds of recombinant lentivirus plasmids for TGF-β1 shRNA were constructed successfully determined by DNA sequencing.The expression of TGF-β1 at mRNA and protein level was decreased among the three kinds of recombinant lentivirus plasmids infected groups compared to the blank control and the negative control,of which TGF-β1 shRNA 2 had the best protein silence efficiency(57%).Conclusion:The recombinant lentiviral vectors for rat TGF-β1 shRNA interference were constructed and the silence efficiency was identified in rat primary detrusor cells,providing a possible basis for further gene therapy research.
作者
赵彦坡
贾春松
ZHAO Yan-po;JIA Chun-song(Department of Neurology,Mentougou District Hospital,Beijing 102300;Department of Urology,Xuanwu Hospital,Capital Medical University,Beijing 100053,China)
出处
《川北医学院学报》
CAS
2020年第2期177-182,共6页
Journal of North Sichuan Medical College
基金
北京市自然科学基金面上项目(7162078)。
作者简介
赵彦坡(1976-),男,硕士,副主任医师。E-mail:zhaoyanpo524@163.com;通讯作者:贾春松,博士。E-mail:jm10301050@126.com。
