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.docVIP

精品论文 参考文献 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 DOI：10.7504/nk2016010203 中图分类号：R587.1 文献标识码：A 　　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Vbeta;13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Vbeta;13 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Vbeta;13的shRNA表达质粒，构建携带Vbeta;13的mCherry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Vbeta;13序列后，将有效的UTG- Vbeta;13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Vbeta;13-shRNA慢病毒载体UTG-shRNA- Vbeta;13，RT-PCR及Western Blot结果显示慢病毒感染的293FT细胞Vbeta;13 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人载体，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。 　　关键词:1型糖尿病，慢病毒，绿色荧光蛋白，红色荧光质粒，Vbeta;13 　　An EGFP-Lentivirus and red target Vbeta;13 gene system and its infection in rat T cells 　　LIUZhi-Jun*1CHENYong2LIWen-Tong3WANGJin-Dong1LIJiao4JIANGZi-Tong5LIFeng-Jie6 　　1.Microbiology Department, Weifang Medical University Shandong Weifang 261053 　　2.Biochemistry Department, Weifang Medical University Shandong Weifang 261053 　　3.Department of Pathology, Weifang Medical University Shandong Weifang 261053 　　4. Biological Science and Technology, Biopharmaceutical class 2015th 　　Weifang Medical University Shandong Weifang 261053 　　5. Clinical Medicine,2014th，Weifang Medical University Shandong Weifang 261053 　　6. Central Laboratory，Weifang Medical University Shandong Weifang 261053 　　Abstract: [Objective] RNA interferience technology mediated by Lentivirus was applied to effectively silence the mCherry combined Vbeta;13 target gene expression and a possibility in Lentivirus transduction NSCs was explored preliminarily [Methods] The Vbeta;13gene mRNA sequences were set as interfered targets and a Lentivirus cassette named UTG was used as vector in producing Lentivirus to construct shRNA expressive plasmid. A mCherry plasmid carrying the whole backbone of Vbeta;13 gene was set as the target to shRNAs. Both the UTG and mCherry- Vbeta;13 pla