| 摘要: |
| 【目的】克隆牛PLIN2和CGI-58基因CDS序列并构建蛋白互作载体，对PLIN2和CGI-58蛋白的互作关系进行验证，为后续研究2种蛋白互作对秦川牛脂肪细胞早期脂滴生成的影响提供试验依据。【方法】PCR扩增牛PLIN2和CGI-58基因CDS区序列，将其分别无缝克隆到线性化pBiFC-VN-173、pBiFC-VC-155载体中，构建蛋白双分子荧光互补载体pBiFC-VN-173-PLIN2、pBiFC-VC-155-PLIN2、pBiFC-VN-173-CGI-58和pBiFC-VC-155-CGI-58。将pBiFC-VN-173-PLIN2＋pBiFC-VC-155-CGI-58，pBiFC-VC-155-PLIN2＋pBiFC-VN-173-CGI-58共转染牛原代脂肪细胞，以空载pBiFC-VN-173＋BiFC-VC-155共转染处理作为阴性对照，用显微镜观察融合蛋白黄色荧光情况，并采用流式细胞仪检测黄色荧光强度。用实时荧光定量RCR法检测PLIN2和CGI-58蛋白双分子荧光互补载体共转染对牛原代脂肪细胞脂质合成相关基因PPARγ、C/EBPα、FASN、ACLY、ACC1表达量的影响。【结果】pBiFC-VN-173-PLIN2＋pBiFC-VC-155-CGI-58共转染牛原代脂肪细胞培养24 h后检测到黄色荧光，且荧光强度极显著高于pBiFC-VN-173-CGI-58＋pBiFC-VC-155-PLIN2共转染组和对照组（P<0.01）；流式细胞仪检测结果显示，pBiFC-VN-173-PLIN2＋pBiFC-VC-155-CGI-58组出现明显的黄色荧光细胞聚类现象，表明牛PLIN2蛋白与CGI-58蛋白存在互作关系。pBiFC-VN-173-PLIN2＋pBiFC-VC-155-CGI-58共转染的牛脂肪细胞脂质合成关键基因表达量与对照组相比未出现明显差异，表明构建的载体未对脂肪细胞生长及脂质合成等生理过程产生影响。【结论】成功构建了牛PLIN2和CGI-58蛋白互作载体，并证明这2种蛋白存在互作关系；构建的载体未对牛脂肪细胞脂质合成关键基因表达量产生明显影响。 |
| 关键词: PLIN2 CGI-58 蛋白互作 脂肪细胞 秦川牛 |
| DOI：10.13207/j.cnki.jnwafu.2025.07.001 |
| 分类号: |
| 基金项目:陕西省重点研发计划项目（2023-YBNY-142）；国家肉牛牦牛产业技术体系建设项目（CARS-37）；国家重点研发计划项目（2018YFD0501700） |
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| Research of PLIN2 and CGI-58 protein-protein interaction in bovine |
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LI Peiwei1, GONG Congran1, SUN Yuting1, ZAN Linsen2
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1.Shaanxi Institute of Zoology,Xi’an，Shaanxi 710032,China;2.College of Animal Science and Technology,Northwest A&F University,Yangling，Shaanxi 712100,China
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| Abstract: |
| 【Objective】The sequence of PLIN2 and CGI-58 CDS of Qinchuan cattle was cloned and the protein-protein interaction (PPI) vector was constructed to verify the interaction between PLIN2 and CGI-58,in order to provide experimental basis for further study on the effect of PLIN2 and CGI-58 PPI on nascent lipid droplet formation in bovine adipocytes.【Method】Bovine of PLIN2 and CGI-58 CDS sequence was amplified and 4 groups of BiFC vectors(pBiFC-VN-173-PLIN2,pBiFC-VC-155-CGI-58,pBiFC-VC-155-PLIN2,pBiFC-VN-173-CGI-58) were constructed by infusion cloning method.Then pBiFC-VN-173-PLIN2+pBiFC-VC-155-CGI-58 and pBiFC-VC-155-PLIN2+pBiFC-VN-173-CGI-58 were transfected into bovine adipocytes.The pBiFC-VN-173＋BiFC-VC-155 empty vectors were also transfected into bovine adipocytes,which served as negative control.The microscope was used to observe the fluorescence,and the flow cytometry was used to detect the fluorescence intensity.The qRT-PCR was also used to detect the effect of PLIN2 and CGI-58 PPI vectors transfection on the expression level of PPARγ、C/EBPα、FASN、ACLY and ACC1 genes,which were related to the lipogenesis in bovine adipocytes.【Result】Yellow fluorescence was detected in bovine adipocytes cotransfected by pBiFC-VN-173-PLIN2＋pBiFC-VC-155-CGI-58 after 24 h of culture,and its fluorescence intensity was significantly higher than that in pBiFC-VN-173-CGI-58＋pBiFC-VC-155-PLIN2 transfected group and negative control group（P<0.01）.The clustering of yellow fluorescence was also detected in pBiFC-VN-173-PLIN2＋pBiFC-VC-155-CGI-58 group by flow cytometry.These results indicated that PLIN2 could interact with CGI-58 in bovine.No significant difference was observed in the expression level of key genes related to lipogenesis between the pBiFC-VN-173-PLIN2＋pBiFC-VC-155-CGI-58 group and the control,indicating no influence of the constructed vectors on the physiological processes of adipocytes lipogenesis.【Conclusion】PLIN2 and CGI-58 PPI vectors were constructed successfully.Bovine PLIN2 and CGI-58 PPI relationship were proved.These vectors showed little influence on bovine adipocytes lipogenesis physiological process(P＞0.05). |
| Key words: PLIN2 CGI-58 protein-protein interaction adipocytes Qinchuan cattle |
