靶向敲除EB病毒潜伏膜蛋白1（LMP1）编码基因重组慢病毒的构建和应用

发布时间：2018-05-26 13:56

本文选题：EB病毒 + 潜伏膜蛋白1　；参考：《青岛大学》2017年硕士论文

【摘要】：目的:探讨LMP1特异性表达沉默对EBV阳性鼻咽癌细胞系C666-1细胞增殖、侵袭转移能力的影响;以及EBV编码基因LMP1对lncRNA H19基因启动子区甲基化和表达的影响。方法:①采用慢病毒感染法分别以MOI值为1,10,100绿色荧光标记的阴性对照病毒sgRNA(U6-SV40-EGFP)接种至C666-1细胞,并分别将4种感染条件加至3种MOI值中,感染72h后采用倒置荧光显微镜观察感染效率。②体外培养鼻咽癌C666-1细胞,加入梯度浓度的嘌呤霉素溶液筛选,确定最佳筛选浓度。③设计并合成2种针对LMP1基因的sgRNA(LV-LMP1-413,LV-LMP1-414)及Cas9嘌呤霉素抗性的慢病毒颗粒。在预实验确认的MOI值和最佳感染条件下,将携带嘌呤霉素标记的Cas9慢病毒颗粒接种至C666-1细胞中,采用预实验确认的嘌呤霉素浓度筛选出稳定表达Cas9的细胞株,然后将携带绿色荧光蛋白与sgRNA(LV-LMP1-413,LV-LMP1-414)的慢病毒分别感染至Cas9蛋白稳转C666-1细胞,同时设病毒对照和细胞对照;采用有限稀释法筛选出同时携带绿色荧光和嘌呤霉素抗性的单克隆细胞,扩大培养并建立稳定细胞系。④采用Western blotting检测靶基因LMP1沉默前后蛋白水平的变化;CCK8细胞增殖实验和transwell小室检测LMP1基因沉默前后对C666-1细胞增殖及侵袭转移能力的影响;亚硫酸盐基因组测序法(bisulfate genomic sequencing,BGS)检测 LMP1 沉默前后 C666-1 细胞系中 lncRNA H19 动子区甲基化程度;实时荧光定量 PCR(Real time fluorescent quantitative PCR,real time qPCR)检测LMP1沉默前后C666-1细胞系中lncRNAH19的转录表达。结果:①对照病毒转染C666-1细胞后,72h在倒置荧光显微镜下观察到细胞内有点状绿色荧光出现,感染效率高于80%,且细胞生长良好,表明转染成功。本实验选用MOI值为100和最佳感染条件(polybrene和ENi.S.液)进行Cas9蛋白和sgRNA转染C666-1细胞。②经筛选确认嘌呤霉素最佳使用浓度为1μg/ml。③成功筛选出同时携带绿色荧光和嘌呤霉素抗性的C666-1细胞克隆。Western blotting结果显示,与阴性病毒对照及细胞对照相比,LMP1沉默后C666-1细胞中LMP1表达明显减弱。④与阴性对照及细胞对照比较,LMP1基因沉默后C666-1细胞增殖、侵袭、迁移能力明显减弱(P0.05)。⑤BGS结果显示LMP1沉默后C666-1细胞系平均甲基化率(Cas9-EGFP-LMP1-413-C666-1,89.9%,Cas9-EGFP-LMP1-414-C666-1,87.4%);阴性病毒对照及细胞对照平均甲基化率分别为(81.8%,84.8%),统计学分析显示LMP1沉默前后C666-1细胞系中H19基因启动子甲基化水平无显著性差异(P0.05)。⑥LMP1沉默前后C666-1细胞系lncRNA H19的转录表达无显著性差异(P0.05)。结论:①成功构建靶向敲减EBV潜伏期基因LMP1的CRISPR/Cas9双载体慢病毒,并筛选出稳定感染的细胞克隆,初步实验结果表明慢病毒转染能有效沉默靶基因LMP1的表达。②EBV潜伏期基因LMP1表达沉默能抑制EBV阳性鼻咽癌C666-1细胞增殖、侵袭迁移能力。③LMP1表达沉默对lncRNA H19启动子区甲基化及转录表达无明显影响,提示尚有其他因素参与EBV对lncRNA H19甲基化和表达的调控作用。
[Abstract]:Objective: To investigate the effect of LMP1 specific expression silencing on the proliferation, invasion and metastasis of EBV positive nasopharyngeal carcinoma cell line C666-1 cells, and the effect of EBV encoding gene LMP1 on the methylation and expression of lncRNA H19 promoter region. GRNA (U6-SV40-EGFP) was inoculated to C666-1 cells, and 4 kinds of infection conditions were added to 3 MOI values. Infection efficiency was observed by inverted fluorescence microscope after 72h infection. (2) C666-1 cells of nasopharyngeal carcinoma were cultured in vitro, and the optimum screening concentration was selected by adding a gradient concentration of purinomycin solution. (3) 2 kinds of sgRN for LMP1 gene were designed and synthesized. A (LV-LMP1-413, LV-LMP1-414) and Cas9 purinomycin resistant lentivirus particles. Under the pre confirmed MOI value and the optimal infection condition, the Cas9 lentivirus particles labeled with purinomycin were inoculated into C666-1 cells, and the cell lines that stably expressed Cas9 were screened by the preconfirmed concentration of purinomycin, and then the Cas9 was carried green. The lentivirus of fluorescent protein and sgRNA (LV-LMP1-413, LV-LMP1-414) infected with Cas9 protein to stabilize C666-1 cells, and also set the virus control and cell control. The monoclonal cells carrying both green fluorescence and purinamycin resistance were screened by the finite dilution method, and Kuo Dapei cultured and established the stable cell line. (4) the Western blotting examination was used. The changes in the protein level of the target gene LMP1 before and after the silencing of the target gene, the effect of the proliferation and invasion and metastasis of C666-1 cells before and after the LMP1 gene silencing by the Transwell cell test and the CCK8 cell proliferation test, and the assay of bisulfate genomic sequencing, BGS to detect the lncRNA zone in the C666-1 cell line of LMP1 before and after the silencing of LMP1 Real-time fluorescence quantitative PCR (Real time fluorescent quantitative PCR, real time qPCR) was used to detect the transcriptional expression of lncRNAH19 in C666-1 cell lines before and after the silencing of LMP1. Results: (1) after transfection of the virus to the cells, the appearance of a dot like green fluorescence in the cell was observed under the inverted fluorescence microscope, and the infection efficiency was higher than 80% The cell growth was good and the transfection was successful. The experiment selected the MOI value of 100 and the best infection condition (Polybrene and ENi.S. liquid) for Cas9 protein and sgRNA transfected C666-1 cells. 2. The optimal use concentration of purinamycin was 1 mu g/ml. (3), and a successful screening of C666-1 cell clone carrying green fluorescence and purinamycin resistance was successfully screened. The results of.Western blotting showed that the LMP1 expression in C666-1 cells was significantly weakened after LMP1 silencing with negative viruses and cell pairs. 4. Compared with negative control and cell control, the proliferation, invasion, and migration ability of C666-1 cells decreased significantly after LMP1 gene silencing (P0.05). 5. BGS results showed that C666-1 cell line methylated by LMP1 after the LMP1 was silent. The rate of Cas9-EGFP-LMP1-413-C666-1,89.9% (Cas9-EGFP-LMP1-414-C666-1,87.4%) and the average methylation rate of negative virus control and cell control were respectively (81.8%, 84.8%). Statistical analysis showed that there was no significant difference in the level of H19 gene promoter methylation in C666-1 cell lines before and after LMP1 silence (P0.05). 6. C666-1 cell line lncRN before and after LMP1 silencing. There was no significant difference in the transcriptional expression of A H19 (P0.05). Conclusion: (1) a successful construction of a CRISPR/Cas9 double vector lentivirus targeting the target EBV latency gene LMP1 was successfully constructed and a stable infection cell clone was screened. The preliminary experimental results showed that the gene expression of the target gene LMP1 could be silenced effectively by the transfection of the lentivirus. (2) the LMP1 expression silencing of the EBV latency gene could inhibit E. BV positive nasopharyngeal carcinoma C666-1 cell proliferation and invasion and migration. LMP1 expression silencing has no obvious effect on the methylation and transcriptional expression of lncRNA H19 promoter region, suggesting that there are other factors involved in the regulatory role of EBV on the methylation and expression of lncRNA H19.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R373

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