摘要
【目的】基于双分子荧光互补(Bimolecular fluorescence complementation, BiFC)技术研究依据猪流行性腹泻病毒(PEDV)S蛋白受体结合位点肽段的靶标肽与互补肽在活细胞内的相互作用关系以及定位情况,为进一步利用该多肽进行猪流行性腹泻病毒的检测积累前期基础和提供理论支撑。【方法】依据靶标蛋白的氨基酸组成、所带电荷种类、形成分子间氢键的能力、极性的强弱、疏水性能等理化特性设计出靶标肽;根据靶标蛋白的氨基酸组成,设计出在理论上与其相互作用的互补肽,并利用生物信息学工具对靶标肽与互补肽进行预测分析;将靶标肽与互补肽的核苷酸序列分别插入EcoRΙ/XhoΙ双酶切后的pBiFC-VC155与NotΙ/SalΙ双酶切后的pBiFC-VN173载体中,构建出BiFC真核表达载体,经酶切及测序验证正确后转染Vero细胞来研究靶标肽与互补肽的互作关系以及形成的BiFC复合物在细胞内的精确定位。【结果】生物信息学分析表明靶标肽和互补肽分别带有亲水性和疏水性结构域,亲水性结构域都带有正负电荷,能够产生静电引力;酶切鉴定和测序结果表明成功构建了pBiFC-VC155-靶标肽和pBiFC-VN173-互补肽真核表达质粒;细胞转染试验表明重组质粒共同转染后靶标肽和互补肽能够在Vero细胞内形成BiFC复合物,表明两者发生了相互作用;间接免疫荧光试验证实该BiFC复合物主要分布于细胞核中。【结论】研究证实了基于猪流行性腹泻病毒S蛋白受体结合位点肽段设计的多肽能够在活细胞内发生相互作用,表明该肽段用于检测的可行性。
【Objective】Using the bimolecular fluorescence complementation(BiFC)technology,the present experiment aimed to study the interaction relationship and localization of the target peptide and the complementary peptide based on the porcine epidemic diarrhea virus(PEDV)S protein receptor binding site peptide in living cells,so as to provide the foundation and theoretical support for the further use of the peptide in the detection of porcine epidemic diarrhea virus.【Method】The target peptide was designed according to the physical and chemical characteristics of the target protein,such as the amino acid composition,the type of charge,the ability to form intermolecular hy-drogen bonds,the strength of polarity,and hydrophobicity;According to the amino acid composition of the target protein,a complementary peptide that interacted with it in theory was designed,and the target peptide and complementary peptide were predicted and analyzed by u-sing bioinformatics tools;The target peptide and complementary peptide were inserted into the p BiFC-VC155 and pBiFC-VN173 vector,which was double digested by the EcoRI/Xho I and Not I/Sal I,respectively,verified by enzyme digestion and sequencing,and then transfect-ed into Vero cells to study the interaction between the target peptide and the complementary peptide,and the precise localization of BiFC complex in cells.【Result】Bioinformatics analysis showed that the target peptide and complementary peptide had hydrophilic and hydrophobic domains,respectively,and the hydrophilic domains were both positively and negatively charged,which could generate electrostatic attrac-tion.The results of enzyme digestion and sequencing showed that the p BiFC-VC155-target peptide and p BiFC-VN173-complementary peptide plasmids were successfully constructed;Cell transfection experiments showed that the target peptide and complementary peptide could form BiFC complexes in Vero cells after co-transfection of recombinant plasmids,indicating that they could interact with each other;Indirect im-munofluorescence assay confirmed that the BiFC complex was mainly distributed in the nucleus.【Conclusion】It was confirmed that the pep-tide designed based on the PEDV S protein receptor binding site can interact with each other in living cells,demonstrating the feasibility of the peptide for detection.
作者
梁洪宇
刘成倩
高骏
周佳明
司伏生
易建中
LIANG Hong-yu;LIU Cheng-qian;GAO Jun;ZHOU Jia-ming;SI Fu-sheng;YI Jian-zhong(Institute of Animal Husbandry and Veterinary Science,Shanghai Academy of Agricultural Sciences,Shanghai 201106,China;Shanghai Jinshan District Animal Center of Disease Control,Shanghai 201500,China)
出处
《西南农业学报》
CSCD
北大核心
2023年第2期427-434,共8页
Southwest China Journal of Agricultural Sciences
基金
国家自然科学基金项目(32072838)。
关键词
猪流行性腹泻病毒
靶标肽
互补肽
蛋白互作
双分子荧光互补
病原诊断
PEDV
Target peptide
Complementary peptide
Protein interaction
Bimolecular fluorescence complementary
Pathogen diag-nosis
作者简介
第一作者:梁洪宇(1997-),女,硕士,研究方向为动物疫病诊断。E-mail:767329172@qq.com;通讯作者:司伏生(1983-),男,博士,副研究员,主要从事动物肠道冠状病毒的致病机制研究。E-mail:mr.fusheng@163.com;通讯作者:易建中(1968-),男,博士,研究员,主要从事动物疫病诊断相关研究。E-mail:yijianzhong@yahoo.com。
