| 奶牛妊娠相关糖蛋白2的表达及结构功能预测分析 |
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| 引用本文： | 杨亚军,何金科,白天鸽,宋胜男,杨宁宁,陈创夫,王震. 奶牛妊娠相关糖蛋白2的表达及结构功能预测分析[J]. 中国畜牧兽医, 2020, 47(10): 3039-3048. DOI: 10.16431/j.cnki.1671-7236.2020.10.001 |
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| 作者姓名： | 杨亚军 何金科 白天鸽 宋胜男 杨宁宁 陈创夫 王震 |
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| 作者单位： | 1. 石河子大学生命科学学院, 石河子 832000;2. 石河子大学动物科技学院, 石河子 832000;3. 石河子大学医学院, 石河子 832000;4. 石河子大学西部地区高发人兽共患传染性疾病防治协同创新中心, 石河子 832000 |
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| 基金项目： | 国家自然科学基金（31572491） |
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| 摘 要： | 为了获得高效表达纯化并且抗原性较好的牛妊娠相关糖蛋白2（bovine pregnancy-associated glycoprotein 2,BoPAG2）,对该蛋白的结构与功能进行生物信息学预测分析。通过PCR扩增荷斯坦奶牛BoPAG2基因,并将该基因连接至表达载体pET-28a,转化至大肠杆菌（BL21）中诱导表达并纯化,通过SDS-PAGE和Western blotting验证与检测该蛋白的大小和免疫学特性,通过生物信息学对BoPAG2蛋白的糖基化修饰、B细胞抗原表位、亲水性、信号肽,蛋白二级结构和3D模型、保守结构域以及蛋白互作进行预测分析。结果表明,本研究成功克隆出BoPAG2基因,通过诱导表达并纯化相应蛋白,通过SDS-PAGE和Western blotting验证BoPAG2蛋白的大小与预期结果相符,通过生物信息学预测分析结果显示,BoPAG2蛋白有5个位点发生糖基化修饰,分别为Asp51、Asp71、Asp114、Asp252和Asp343,该蛋白含有15个B细胞抗原表位,在N端含有15个氨基酸组成的信号肽序列,二级结构主要以无规则卷曲和延伸链为主,并且成功建立了BoPAG2蛋白的3D模型,该蛋白含有4个超家族结构域,蛋白互作分析结果显示,与BoPAG2互作的蛋白有7个,该蛋白可能参与了消化和吸收,以及母体在妊娠期的调节功能等过程。综上所述,本试验成功表达并纯化获得了BoPAG2蛋白。通过对BoPAG2蛋白的结构及功能进行生物信息学预测分析,为BoPAG2蛋白抗体的制备以及功能的研究提供了理论基础。
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| 关 键 词： | 妊娠相关糖蛋白 原核表达 抗原表位 信号肽 |
Expression and Structure Function Prediction of Bovine Pregnancy-associated Glycoprotein 2 |
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| YANG Yajun,HE Jinke,BAI Tiange,SONG Shengnan,YANG Ningning,CHEN Chuangfu,WANG Zhen. Expression and Structure Function Prediction of Bovine Pregnancy-associated Glycoprotein 2[J]. China Animal Husbandry & Veterinary Medicine, 2020, 47(10): 3039-3048. DOI: 10.16431/j.cnki.1671-7236.2020.10.001 |
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| Authors: | YANG Yajun HE Jinke BAI Tiange SONG Shengnan YANG Ningning CHEN Chuangfu WANG Zhen |
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| Affiliation: | 1. College of Life Sciences, Shihezi University, Shihezi 832000, China;2. College of Animal Science and Technology, Shihezi University, Shihezi 832000, China;3. Medical College, Shihezi University, Shihezi 832000, China;4. Collaborative Innovation Center for the Prevention and Control of Communicable Diseases in Western China, Shihezi University, Shihezi 832000, China |
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| Abstract: | In order to obtain bovine pregnancy-associated glycoprotein 2 (BoPAG2) with high expression and purification and good antigenicity,the structure and function of the protein were analyzed by bioinformatics prediction.The BoPAG2 gene of Holstein dairy cow was amplified by PCR,and the gene was ligated into the expression vector pET-28a,transformed into E.coli (BL21) to induce expression and purification.SDS-PAGE and Western blotting were used to verify and detect the size and immunological characteristics of the protein.The glycosylation modification,B-cell epitope,hydrophilicity,signal peptide,secondary structure and 3D model,conservative domain and protein interaction of BoPAG2 protein were predicted and analyzed by bioinformatics.The results showed that the BoPAG2 gene was successfully cloned,and the corresponding protein was expressed and purified by induction.The size of the BoPAG2 protein was consistent with the expected results by SDS-PAGE and Western blotting.The results of bioinformatics prediction analysis showed that five sites of BoPAG2 protein occurred glycosylation modification,Asp51,Asp71,Asp114,Asp252 and Asp343,respectively.The protein contained 15 B-cell epitopes and a signal peptide sequence consisting of 15 amino acids in the N terminal.The 3D model of the BoPAG2 protein was successfully established.The protein contained 4 superfamily domains.The results of protein interaction analysis showed that there were 7 proteins that interact with BoPAG2.The protein might be involved in digestion and absorption.Other processes in the maternal regulatory function during pregnancy.In summary,the BoPAG2 protein was successfully expressed and purified in this experiment.The bioinformatics prediction analysis of the structure and function of BoPAG2 protein provided a theoretical basis for the preparation and function research of BoPAG2 protein antibody. |
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| Keywords: | pregnancy-associated glycoproteins prokaryotic expression antigen epitopes signal peptide |
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