PEDV E蛋白的跨膜结构域对其细胞定位的影响研究  

Effect of Transmembrane Domain of PEDV Envelope Protein on Cellular Localization

作　　者：郭德轩 郑亮 张宁 郑小会 吴志军[1,2,3] 曹宏伟 张华[1,2,3] Guo Dexuan;Zheng Liang;Zhang Ning;Zheng Xiaohui;Wu Zhijun;Cao Hongwei;Zhang Hua(College of Life Science and Biotechnology,Heilongjiang Bayi Agricultural University,Daqing 163319;College of Animal Science and Technology,Heilongjiang Bayi Agricultural University;Biotechnology Center,Heilongjiang Bayi Agricultural University)

机构地区：[1]黑龙江八一农垦大学生命科学技术学院,大庆163319 [2]黑龙江八一农垦大学动物科技学院 [3]黑龙江八一农垦大学生物技术中心

出　　处：《黑龙江八一农垦大学学报》2022年第4期87-92,共6页journal of heilongjiang bayi agricultural university

基　　金：黑龙江省自然科学基金联合引导项目(LH2020C082)。

摘　　要：猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)为猪流行性腹泻的病原体,感染上该病毒的仔猪出现脱水、腹泻和呕吐,给世界养猪业造成了经济损失。PEDV囊膜蛋白E在病毒包膜形成、病毒离子形成、病毒出芽和释放过程中发挥至关重要的作用。根据软件预测可知,PEDV E蛋白是一种有一个跨膜域的跨膜蛋白。研究将PEDV E蛋白设计好的截短基因(E-T1、E-T2和E-T3)连接至pCMV-myc真核表达载体中,经PCR和双酶切鉴定结果表明已构建成功。最后,将重组质粒pCMV-myc-E、E蛋白截短基因质粒Myc-E-T1、Myc-E-T2和Myc-E-T3转染HEK-293T细胞和IPEC-J2细胞,通过间接免疫荧光技术观察其E蛋白以及截短基因的细胞分布情况,并探究是否与内质网发生共定位。研究结果发现,E蛋白以及E-T1、ET2分布在细胞质并定位在内质网,而E-T3则分布在整个细胞且没有与内质网共定位。说明了E-T1、E-T2可能行使着与E蛋白类似的功能,而E-T3则没有。研究为进一步探讨PEDV的E蛋白功能和分子生物学特性奠定了基础。Porcine epidemic diarrhea virus(PEDV)was the causative agent of porcine epidemic diarrhea.Piglets infected with the virus had dehydration,diarrhea and vomiting,causing economic losses to the pig industry.PEDV envelope protein E played a vital role in virus envelope formation,virus ion formation,virus budding and release.According to software prediction,PEDV E protein was a transmembrane protein with a transmembrane domain.The truncated genes(E-T1,E-T2 and E-T3)designed for PEDV E protein were connected to the pCMV-myc eukaryotic expression vector.The results of PCR and double enzyme digestion showed that the construction was successful.Finally,the recombinant plasmid pCMV-myc-E and the constructed E protein truncated gene plasmids Myc-E-T1,Myc-E-T2 and Myc-E-T3 were transfected into HEK-293T cells and IPEC-J2 cells,and then immunized indirectly Fluorescence technology observed the nuclear distribution of its E protein and truncated genes,and explored whether it co-localizes with the endoplasmic reticulum.The results found that E protein and E-T1 and E-T2 were distributed in the cytoplasm and localized in the endoplasmic reticulum,while E-T3 was distributed throughout the cell and was not co-localized with the endoplasmic reticulum.It showed that except E-T3,E-T1 and E-T2 might perform functions similar to E protein.It laid a foundation for further exploring the E protein function and molecular biological characteristics of PEDV.

关 键 词：猪流行性腹泻病毒 E蛋白 跨膜结构域 内质网 细胞定位 

分 类 号：R284.1[医药卫生—中药学]