牛支原体脂质相关膜蛋白GLCP抑制宿主EBL细胞凋亡的分子机制研究  

The molecular mechanism of Mycoplasma bovis lipid-associated membrane protein GLCP inhibiting host EBL cells apoptosis

作　　者：张玉娟 刘桐 武琪 徐青元[1] 辛九庆[1] 潘巧 ZHANG Yu-juan;LIU Tong;WU Qi;XU Qing-yuan;XIN Jiu-qing;PAN Qiao(State Key Laboratory for Animal Disease Control and Prevention,Harbin Veterinary Research Institute,Chinese Academy of Agriculture Science,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所动物疫病防控全国重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2023年第6期583-591,共9页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金面上项目(31872468)。

摘　　要：为探究牛支原体(M.bovis)脂质相关膜蛋白(LAMPs)抑制宿主细胞凋亡的分子机制,本研究利用实验室前期经AKTA分子筛分成5组的M.bovis TJ株LAMPs分别刺激胎牛肺(EBL)细胞,经western blot检测各组LAMPs对EBL细胞中凋亡执行分子Caspase-3剪切激活水平的影响,经CCK-8试验检测筛选出的目标LAMPs对EBL细胞活力的影响,并对其进行蛋白质谱测序分析。结果显示,D组LAMPs能够抑制Caspase-3的剪切激活水平,并可以极显著提高EBL细胞活力,结合蛋白质谱测序结果以及生物信息学软件预测蛋白功能,选定VSPHB0801-4、TPP、DJ-1、EF-TU、GAPDH以及GLCP为候选蛋白进行后续试验。经PCR扩增dj-1基因,重叠延伸PCR扩增ef-tu和glcp融合基因,vsphb0801-4、tpp和gapdh基因片段则由华大基因公司合成,将上述基因片段分别克隆至相应的原核表达载体中,构建重组质粒pET-28a-vsphb0801-4、pET-28a-tpp、pET-32a-dj-1、pET-32a-ef-tu、pEGX-6P-1-gapdh和pMAL-c5X-glcp,并分别转化大肠杆菌BL21(DE3),经IPTG诱导后通过亲和层析纯化目的蛋白,SDS-PAGE检测重组蛋白的纯化效果。结果显示,获得了纯度较高的6个重组蛋白。将不同浓度的6个重组蛋白分别刺激EBL细胞,经western blot验证重组蛋白是否具有抑制EBL细胞凋亡的作用,结果显示,只有重组蛋白GLCP能够抑制EBL细胞凋亡。因此,将其作为靶标蛋白进一步探究其抑制EBL细胞凋亡的分子机制。将不同浓度的重组蛋白GLCP分别刺激EBL细胞后,经western blot检测经典凋亡通路介导分子Caspase-8、Caspase-9和Caspase-12的剪切激活水平、激光共聚焦显微镜检测EBL细胞线粒体膜电位,以及western blot检测线粒体介导的凋亡通路关键分子Bcl-2、Bax、Cyt C、Apaf-1以及剪切的多聚ADP核糖聚合酶-1(PARP1)蛋白的表达水平。结果显示,重组蛋白GLCP主要抑制Caspase-9的剪切激活水平,提高线粒体膜电位,并上调线粒体介导的凋亡通路关键分子Bcl-2蛋白的�To establish a LAMP method for rapid detection of Neospora caninum.In this study,two pairs of specific primers were designed based on the Nc-5 gene sequence(X84238.1)of Neospora caninum registered in GenBank,and the amplified sequence of Nc-5 gene was then cloned into the pMD18-T vector,resulting a recombinant plasmid pMD18-T-Nc-5.After PCR and sequencing identification,pMD18-T-Nc-5 was used as the standard plasmid in the following experiment and its concentration was determined using a UV spectrophotometer and converted to copy number through a formula.The primer concentration and reaction temperature of the method were optimized,and the turbidity value of magnesium pyrophosphate was monitored in real time when the target gene was amplified by loop-mediated isothermal amplification(LAMP)turbidity meter,thus the LAMP method for detecting Neospora caninum was preliminarily established.After the reaction,the results were visualized and determined by adding SYBR Green I.The results of optimization reaction conditions showed that the final concentrations of primer F3/B3 and FIP/BIP were 10pmol/μL and 20pmol/μL,respectively,and the optimal reaction temperature was 63℃and amplification for 40 minutes.This method was used to detect the nucleic acid of Neospora caninum,Toxoplasma gondii,Theileria sergenti,Theileria annulata,Batesiosis Bigeminum and Trypanosoma evansi.The results showed that only the amplification of Neospora caninum was positive,and the rest of the pathogenic nucleic acids were negative.The fluorescence observation results showed that the reaction tubes of Neospora caninum was emerald green fluorescence(positive),and the other reaction tubes were orange yellow fluorescence(negative).The sensitivity test was performed on the recombinant plasmid standards pMD18-T-Nc-5 after 10-fold dilution as a template.The LAMP amplification results showed that the detection limit of this method for plasmid standards was 4.0×10^(1) copies/μL.After the reaction was completed,when SYBR Green I was added,the reaction

关 键 词：牛支原体 细胞凋亡 脂质相关膜蛋白 线粒体介导的凋亡通路 

分 类 号：S852.62[农业科学—基础兽医学]