论文部分内容阅读
目的构建靶向MA104细胞多聚嘧啶序列结合蛋白(polypyrimidine tract-binding protein,PTB)的shRNA慢病毒干扰质粒,并对其进行鉴定。方法根据Gen Bank中登录的PTB的基因序列设计并合成2条靶向MA104细胞PTB基因的shRNA干扰序列(shPTB1、shPTB2)和1条阴性对照序列(shControl),分别插入载体p LVshRNA-EGFP(2A)Puro后,构建慢病毒重组质粒;利用HEK293Ta细胞包装针对PTB基因的shRNA的重组慢病毒颗粒;感染MA104细胞后,经Resl-time PCR、免疫荧光和Western blot法分别检测MA104细胞中PTB基因的转录和蛋白表达水平。结果 PCR及测序鉴定证明3种重组慢病毒质粒构建正确。3种重组慢病毒质粒转染HEK293Ta细胞48 h后,均可见绿色荧光表达,3组细胞的病毒滴度均为2×106 TU/ml。3种重组慢病毒感染MA104细胞48 h后均可见绿色荧光表达。经嘌呤霉素加压筛选后,p LVshPTB1-EGFP-2A和p LVshPTB2-EGFP-2A组MA104细胞仅有少量细胞于细胞核中表达,p LVshPTB1-EGFP-2A组MA104细胞中PTB基因转录及蛋白表达的水平均低于p LVshPTB2-EGFP-2A组。结论成功构建了靶向MA104细胞PTB蛋白的shRNA重组慢病毒,shRNA序列可成功下调PTB在MA104细胞中的表达,为PTB在RV增殖过程中调控作用的研究奠定了基础。
Objective To construct a shRNA lentivirus plasmid targeting the polypyrimidine tract-binding protein (PTB) of MA104 cells. Methods shRNA interference sequences (shPTB1, shPTB2) and a negative control sequence (shControl) of two PTB genes targeting MA104 cells were designed and synthesized according to the gene sequence of PTB registered in GenBank and inserted into vector p LVshRNA-EGFP (2A ) Puro was constructed, and the recombinant lentiviral plasmid was constructed by using HEK293Ta cells. The recombinant lentivirus particles targeting PTB gene shRNA were packaged by HEK293Ta cells. After infected with MA104 cells, the expression of PTB gene in MA104 cells was detected by Resl-time PCR, immunofluorescence and Western blot Transcription and protein expression levels. Results PCR and sequencing showed that the three recombinant lentiviral plasmids were constructed correctly. The HEK293Ta cells transfected with the three kinds of recombinant lentiviral plasmids were transfected with HEK293Ta cells for 48 hours, and the green fluorescence was observed. The virus titer of the three groups was 2 × 106 TU / ml. The three recombinant lentivirus-infected MA104 cells were observed after 48 h of green fluorescent expression. Only a small number of cells were expressed in the nucleus of MA104 cells in p LVshPTB1-EGFP-2A and p LVshPTB2-EGFP-2A groups by pURshPTB1-EGFP-2A and PTB gene transcription and protein expression in MA104 cells in p LVshPTB1-EGFP-2A group Were lower than the p LVshPTB2-EGFP-2A group. Conclusion shRNA recombinant lentivirus targeting PTB protein of MA104 cells was constructed successfully. ShRNA sequence can down-regulate the expression of PTB in MA104 cells, which lays the foundation for the research of PTB in the regulation of RV proliferation.