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。

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。

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。

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。

Construction of the mmu_circ_0001033 lentiviral expression vector as the basis for the study of its functional experimental study in hypoxic pulmonary hypertension.

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.

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.

The mmu_circ_0001033 lentiviral expression vector was successfully constructed, which can stably transfect 293T cells and can be used for subsequent cell experiments.