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摘要：环状RNA分子（Circular RNAs，circRNAs）是一种广泛存在于哺乳动物细胞中特殊的非编码RNA分子，是目前关于circRNAs的研究是RNA研究领域的热点之一。本课题组前期通过高通量测序筛选得到一个丝/苏氨酸蛋白激酶(serine-threoninekinase，Akt)基因外显子形成的一条circRNA，命名为Circ-AKT2。本研究通过PCR扩增得该circRNA全长，构建慢病毒重组表达载体，通过共转染和筛选成功获得过表达Circ-AKT2细胞系。利用此细胞系研究Circ-AKT2在流感病毒A/PR/8毒株侵袭过程中可能发挥的作用及调控机制，为流感病毒的防控提供了新的思路。28595
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毕业论文关键词：Circ-AKT2；过表达细胞系；流感病毒
Overexpressing Circ-AKT2 cell line construction and Preliminary study on Circ-AKT2 function
Abstract：Circular RNAs (circRNAs) are a novel type of RNA that, unlike linear RNAs, form a covalently closed continuous loop and are highly represented in the eukaryotic transcriptome.The study of circRNAs has gradually become one of the most noticeable areas in the field of RNA biology.This study using previous high-throughput sequencing screen for a circRNA formed by exons of serine/threonine protein kinase(serine-threoninekinase,Akt)genes named Circ-AKT2.The full-length circRNA was amplified by PCR.To construct lentiviral recombinant expression vector, the Circ-AKT2 cell line was successfully obtained by co-transfection and screeningdesigned primers.This cell line is used to investigate the possible role of Circ-AKT2 in the invasion of influenza virus A/PR/8 strain and its new regulation mechanism, and to provide a new approach for the control of influenza viruses.
Key words:Circ-AKT2; overexpressing cell line；influenza viruses
目录
摘要 3
关键词 3
Abstract 3
Key words 3
引言 3
1 材料与方法 4
1.1材料 4
1.1.1细胞和试剂 4
1.1.2主要仪器 4
1.2实验方法 5
1.2.1细胞培养 5
1.2.1.1 HEK293T细胞培养 5
1.2.1.2 A549细胞培养 5
1.2.2 HA效价和TCID50的测定 5
1.2.2.1 HA效价测定 5
1.2.2.2 TCID50测定 5
1.2.3 A549细胞接A/PR/8（H1N1)毒株 5
1.2.4 RNA提取 5
1.2.5 RNA反转录 5
1.2.5.1 RNA产品用量
1.2.5.2 RNA反转录 5
1.2.6 circ-AKT2检测 6
1.2.6.1检测AKT可能存在的成环位点 6
1.2.6.2配制SYBR Green I实时定量PCR的反应体系 6
1.2.7过表达载体构建 6
1.2.7.1设计过表达载体构建引物 6
1.2.7.2 构建circ-AKT2慢病毒过表达载体 7
1.2.7.2.1 circ-AKT2全长扩增 7
1.2.7.2.2 DNA片段的纯化回收 7
1.2.7.2.3回收产物连接PCDH-ciR载体 7
1.2.8质粒纯化提取 7
1.2.8.1选用抑制重组的菌株Stbl3制备感受态细胞 7
1.2.8.1.1配置溶液 7
1.2.8.1.2用氯化钙制备新鲜的Stbl3感受态细胞 8
1.2.8.2 将circ-AKT2慢病毒过表达载体进行转化 8
1.2.8.3通过PCR鉴定转化子并对阳性克隆测序 8
1.2.8.4去内毒素高纯度质粒抽提 8
1.2.9转染和收集病毒上清 9
1.2.10 转导靶细胞 9
1.2.11 荧光倒置显微镜观察细胞样品荧光 9
2 实验结果 9