摘要
目的:构建牛miR-193a过表达和抑制的慢病毒并进行初步鉴定。方法:根据miRBase数据库中牛miR-193a的前体序列设计引物;以MDBK细胞全基因组为模版,扩增pre-miR-193a和pre-miR-193a inhibitor基因;并克隆至慢病毒载体pLL3.7中;经酶切和测序鉴定后获得重组慢病毒载体;将重组慢病毒质粒分别与辅助质粒共转染至HEK-293T细胞中;转染48h后,收集慢病毒并感染MDBK细胞;感染48 h后,观察MDBK细胞中绿色荧光的分布情况,并收集细胞,提取总RNA并合成cDNA,实时定量PCR检测miR-193a表达水平。结果:成功构建了过表达和抑制miR-193a表达的慢病毒;pre-miR-193a慢病毒感染后miR-193a的表达水平显著性增加(约4.55倍);相反,pre-miR-193a inhibitor慢病毒感染后miR-193a表达水平显著性下调(约0.73倍)。结论:该研究成功构建了牛miR-193a过表达和抑制表达的慢病毒,为进一步的miR-193a的功能学研究奠定基础。
Objective： To construct and preliminary identify the lentiviruses overexpressing and inhibiting Bos taurus miR - 193a. Method： Primers for miR - 193a and miR - 193a inhibitor were designed according to Bos taurus miR - 193a precursor sequence in miRBase database. The genes of pre - miR - 193a and pre - miR - 193a inhibitor were amplified from the genome of MDBK cells and cloned into a lentiviral vector pLL3. 7. The recombinant lentiviral plasmids were identified by double enzyme digestion and sequencing analysis, following by cotransfeetion into HEK -293T cells with the helper plasmids. At 48 hours post - transfection, lentiviral particles were harvested and used to infect MDBK ceils. At 48 hours post - infection, the distribution of green fluorescence in MDBK was observed under an inverted fluorescent microscope. Meanwhile, the cells were collected and subjected to total RNA extraction and cDNA synthesis, following by miR -193a expression levels detection by real -time quantitative PCR. Result ：Lentiviruses overexpressing and inhibiting miR -193a expression were constructed. Moreover, pre - miR - 193a - lentivirus infection significantly upregulated miR - 193a mRNA levels （ About 4. 55 -fold change）. In contrast, miR- 193a expression levels were significantly downregulated in pre- miR- 193a inhibitor- lentivirus infected cells compared with the negative control cells （About 0. 73 -fold change）. Conclusion： Lentiviruses overexpressing and inhibiting Bos taurus miR - 193a were successfully constructed, which will be benefit to further analyze the functions of Bos taurus miR - 193a.
出处
《生物技术》
CAS
CSCD
北大核心
2014年第3期52-57,共6页
Biotechnology
基金
国家国际科技合作专项项目("牛病毒性腹泻防治新技术的研究"
No.2013DFR30970)
国家自然科学基金项目("牛病毒性腹泻病毒持续性感染的分子机制研究"
No.U1303283)资助~~
