|本期目录/Table of Contents|

[1]黄朝旺,韦雅琴,许发琼,等.mmu_circ_0001033过表达慢病毒载体的构建与鉴定[J].中华肺部疾病杂志,2022,(02):141-145.[doi:10.3877/cma.j.issn.1674-6902.2022.02.001]
　Huang Chaowang,Wei Yaqin,et al.Construction and identification of mmu_circ_0001033 overexpression lentiviral vector[J].,2022,(02):141-145.[doi:10.3877/cma.j.issn.1674-6902.2022.02.001]
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mmu_circ_0001033过表达慢病毒载体的构建与鉴定(PDF)

《中华肺部疾病杂志》[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2022年02期
页码:
141-145
栏目:
论著
出版日期:
2022-04-20

文章信息/Info

Title:
Construction and identification of mmu_circ_0001033 overexpression lentiviral vector
作者:
黄朝旺1; 3; 韦雅琴2; 许发琼3; 徐康乔2; 胡 巧1; 张 静1; 夏世金2; 胡明冬1
400037 重庆,陆军(第三)军医大学第二附属医院老年与特勤医学科1、呼吸与危重症医学中心3 200040 上海,复旦大学附属华东医院上海市老年医学研究所2
Author(s):
Huang Chaowang1;  3;  Wei Yaqin2;  Xu Faqiong3;  Xu Kangqiao2;  Hu Qiao1;  Zhang Jing1;  Xia Shijin2;  Hu Mingdong1.
1Department of Geriatrics and Special Services Medicine, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China; 2Institute of Gerontology, Huadong Hospital, Fudan University, Shanghai 200040, China; 3Institute of Respiratory Diseases, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China
关键词:
mmu_circ_0001033;  环状RNA;  慢病毒;  肺动脉高压
Keywords:
Mmu_circ_0001033;  Circular RNA;  Lentivirus;  Pulmonary hypertension
分类号:
R563
DOI:
10.3877/cma.j.issn.1674-6902.2022.02.001
摘要:
目的 构建环状RNA mmu_circ_0001033慢病毒表达载体,作为在低氧性肺动脉高压功能学实验研究的基础。方法 首先设计引物将目的基因片段从原质粒上扩增下来,通过引物两端所含无缝克隆识别位点将其重组到酶切后的过表达载体上; 将连接产物转入制备好的细菌感受态细胞,而后将其单克隆菌落送测序公司进行测序鉴定。其次通过PCR产物跑胶和测序验证成环情况。最后用构建的重组表达载体和包装质粒共同转染293T细胞,包装慢病毒,收集病毒原液,超滤浓缩,并测定滴度。结果 经过比对,重组克隆中插入片段序列与目的片段序列完全一致,表明质粒构建成功。PCR产物后经sanger测序确认环化位点处序列完全正确。将构建的过表达载体测序结果和质粒构建方案基因比对,重组克隆中插入片段序列与目的片段序列与预期完全一致,可实现转染质粒后目的基因的表达,PCR产物跑胶和测序结果表明mmu_circ_0001033成环成功。PCR产物跑胶和测序结果表明mmu_circ_0001033成环成功。最后通过比较RT-qPCR测定病毒滴度值为2.09×108 TU/ml。 结论 成功构建环状RNA mmu_circ_0001033慢病毒表达载体,能稳定转染293T细胞,可用于后续细胞实验。
Abstract:
Objective Construction of the mmu_circ_0001033 lentiviral expression vector as the basis for the study of its functional experimental study in hypoxic pulmonary hypertension. Methods First, primers were designed to amplify the target gene fragment from the original plasmid, and recombined it into the overexpression vector after digestion through the seamless clone recognition sites contained at both ends of the primer, transferred the ligation product into the prepared bacterial sensation Then sent its monoclonal colony to a sequencing company for sequencing and identification. The looping condition was verified by running the gel and sequencing the PCR product. Finally, the constructed recombinant expression vector and packaging plasmid were used to co-transfect 293T cells, packaged the lentivirus, collected the virus stock, concentrated by ultrafiltration, and determined the titer. Results The sequence of the inserted fragment in the recombinant clone was exactly the same as the sequence of the target fragment, indicating that the plasmid was constructed successfully. The PCR product was confirmed by sanger sequencing to confirm the correct sequence at the cyclarization site. By comparing the sequencing results of the constructed overexpression vector and the plasmid construction scheme, the insert sequence and the target fragment sequence in the recombinant clone are exactly as expected, which can realize the expression of the target gene after transfection of the plasmid, the PCR product running gel and sequencing results showed that mmu_circ_0001033 was successfully looped, the PCR product running gel and sequencing results showed that mmu_circ_0001033 was successfully looped. Finally, the virus titer value determined by RT-qPCR was 2.09×108 TU/ml. Conclusion The mmu_circ_0001033 lentiviral expression vector was successfully constructed, which can stably transfect 293T cells and can be used for subsequent cell experiments.

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备注/Memo

备注/Memo:
基金项目: 国家自然科学基金面上项目(81870044,81873421); 高校基础研究课题(2020-2017-075) 通信作者: 胡明冬, Email: huhanshandd@163.com 夏世金, Email: xiashijinhd@163.com
更新日期/Last Update: 2022-04-20