南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (7): 1013-1018.doi: 10.12122/j.issn.1673-4254.2022.07.07

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表达工程化剪接因子腺病毒的构建及其在调控乳鼠心肌细胞中的YAP1可变剪接中的应用

李 阳，赵 倩，宋晓伟，宋金超   

  1. 上海理工大学健康科学与工程学院，上海 200082；上海理工大学附属市东医院麻醉科，上海 200082；海军军医大学第一附属医院心血管内科，上海 200082
  • 出版日期:2022-07-20 发布日期:2022-07-15

Construction of an adenovirus vector expressing engineered splicing factor for regulating alternative splicing of YAP1 in neonatal rat cardiomyocytes

LI Yang, ZHAO Qian, SONG Xiaowei, SONG Jinchao   

  1. School of Health Science and Engineering, University of Shanghai for Science and Technology, Shanghai 200082,China; Department of Anesthesiology, Shidong Hospital Affiliated to University of Shanghai for Science and Technology, Shanghai 200082, China; Department of Heart Medicine, Changhai Hospital, Naval Medical University, Shanghai 200082, China
  • Online:2022-07-20 Published:2022-07-15

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摘要： 目的 构建人工剪接因子，实现调控心肌细胞YAP1的可变剪接。方法 本研究根据Pumilio1的序列特异性，针对YAP1的Exon6构建不同序列的剪接因子。实验分为3组：野生型PUF-SR作为阴性对照组、人工化的PUF-SR腺病毒转染组、质粒转染组。实验流程：使用同源重组的方法构建腺病毒，将人工化的PUF-SR的PCR片段克隆到pAd-Track质粒中，再将重组质粒转化到大肠杆菌DH5α中进行质粒扩增。扩增后的质粒经PacⅠ酶切后，转染到293A细胞，进行腺病毒包装。将得到的腺病毒载体转染到培育的乳鼠心肌细胞中。选用Ad-Track质粒用于对照转染组，通过载体转染法将其转染到乳鼠心肌细胞中。定量和半定量PCR法检测YAP1的可变剪接，Western blot法检测融合蛋白Flag的信号，并使用GAPDH抗体检测GAPDH作为加载对照。结果 腺病毒转染组对心肌细胞的转染效率接近100%，质粒转染组中并没有发现荧光蛋白。在Western blot实验中，阴性对照和靶向YAPExon6XULIE 序列的Flag-SR-NLS-PUF都能够检测到融合Flag的蛋白的表达。在反转录PCR和PCR检测YAP1的可变剪接实验中，结果都显示YAP1的第4个target序列所构建的人工剪接因子能够有效调控YAP1 Exon 6的剪入（P<0.05）。结论 本实验成功构建出能够调控YAP1可变剪接的腺病毒，并且在SD大鼠的心肌细胞中得到了验证。

关键词: 工程化剪接因子；腺病毒；YAP1；可变剪接

Abstract: Objective To construct an adenovirus vector expressing artificial splicing factor capable of regulating alternative splicing of Yap1 in cardiomyocytes. Methods The splicing factors with different sequences were constructed against Exon6 of YAP1 based on the sequence specificity of Pumilio1. The PCR fragment of the artificially synthesized PUF-SR or wild-type PUF-SR was cloned into pAd-Track plasmid, and the recombinant plasmids were transformed into E. coli DH5α for plasmid amplification. The amplified plasmids were digested with Pac I and transfected into 293A cells for packaging to obtain the adenovirus vectors. Cultured neonatal rat cardiomyocytes were transfected with the adenoviral vectors, and alternative splicing of YAP1 was detected using quantitative and semi-quantitative PCR; Western blotting was performed to detect the signal of the fusion protein Flag. Results The transfection efficiency of the adenovirus vectors was close to 100% in rat cardiomyocytes, and no fluorescent protein was detected in the cells with plasmid transfection. The results of Western blotting showed that both the negative control and Flag-SR-NLS-PUF targeting the YAPExon6XULIE sequence were capable of detecting the expression of the protein fused to Flag. The results of reverse transcription-PCR and PCR demonstrated that the artificial splicing factor constructed based on the 4th target sequence of YAP1 effectively regulated the splicing of YAP1 Exon6 in the cardiomyocytes (P<0.05). Conclusion We successfully constructed adenovirus vectors capable of regulating YAP1 alternative splicing rat cardiomyocytes.

Key words: engineered splicing factor; adenovirus; YAP1; alternative splicing