针对糖尿病易感基因Vβ13的慢病毒系统的建立及其对T细胞的转导

发布时间：2018-08-03 17:14

【摘要】：目的应用慢病毒介导的RNA干扰技术,有效沉默连接红色荧光蛋白mCherry与靶基因1型糖尿病易感基因Vβ13的表达,建立一种简便有效基因沉默的系统,并初步研究慢病毒感染T细胞的可能性。方法以Vβ13 mRNA编码序列作为干扰靶点,以慢病毒质粒UTG作为载体,构建靶向Vβ13的shRNA表达质粒,构建携带Vβ13的m Cherry红色荧光蛋白的质粒作为验证shRNA有效性的靶点,并转染293FT细胞。通过红色荧光的表达及RT-PCR和Western blot确定最佳沉默Vβ13序列后,将有效的UTG-Vβ13质粒与辅助质粒共转染293FT细胞,低温超高速离心,获取高滴度慢病毒。应用慢病毒感染大鼠T细胞,光镜观察T细胞被慢病毒感染的情况,流式细胞术检测T细胞的绿色荧光表达情况。结果成功构建Vβ13-shRNA慢病毒载体UTG-shRNA-Vβ13,RT-PCR及Western blot结果显示慢病毒感染的293FT细胞Vβ13 mRNA及蛋白表达相对于对照组显著降低。构建的慢病毒可以成功感染T细胞。结论红色靶基因-绿色慢病毒系统具有简便的筛选性能;UTG慢病毒对神经干细胞具有较高感染效率。可作为一种良好的基因导入载体,实现慢病毒介导的RNA干扰在T细胞内的有效表达,并为T细胞过继性转移的相关研究提供技术支持。
[Abstract]:Objective to effectively silence the expression of red fluorescent protein mCherry and target gene type 1 diabetes susceptibility gene V 尾 13 by lentivirus-mediated RNA interference, and to establish a simple and effective gene silencing system. The possibility of lentivirus infection with T cells was studied. Methods V 尾 13 mRNA coding sequence was used as interference target, lentivirus plasmid UTG was used as vector to construct shRNA expression plasmid targeting V 尾 13, and m Cherry red fluorescent protein carrying V 尾 13 was constructed as the target to verify the validity of shRNA, and 293FT cells were transfected. After the best silencing V 尾 13 sequence was determined by red fluorescence expression and RT-PCR and Western blot, the effective UTG-V 尾 13 plasmid was co-transfected into 293FT cells and centrifuged at low temperature and high speed to obtain high titer lentivirus. Rat T cells were infected with lentivirus. Light microscopy was used to observe the infection of T cells by lentivirus. Flow cytometry was used to detect the green fluorescence expression of T cells. Results RT-PCR and Western blot of V 尾 13-shRNA lentivirus vector UTG-shRNA-V 尾 13 showed that the expression of V 尾 13 mRNA and protein in 293FT cells infected by lentivirus was significantly lower than that in control group. The constructed lentivirus can successfully infect T cells. Conclusion the red target gene-green lentivirus system has a simple screening performance. UTG lentivirus has a high infection efficiency on neural stem cells. It can be used as a good gene transfer vector to realize the effective expression of lentivirus-mediated RNA interference in T cells and provide technical support for the study of T cell adoptive transfer.
【作者单位】：潍坊医学院病原生物学教研室;潍坊医学院病理学教研室;潍坊医学院生物科学与技术学院;
【基金】：国家自然科学基金资助项目(编号:81471048) 山东省自然科学基金资助项目(编号:ZR2012HL42,ZR2015HL064) 山东省高等学校科技计划项目(编号:J12LK01) 潍坊医学院大学生科技创新基金项目(编号:KX2016027)
【分类号】：R587.1

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