牛妊娠相关蛋白19真核表达载体的构建及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2022.04.004

摘要/Abstract

摘要： 【目的】扩增牛妊娠相关蛋白19(bovine pregnancy-associated glycoprotein 19,BoPAG19)基因,构建真核表达载体,并检测其在HEK-293F细胞中的表达。【方法】根据BoPAG19基因序列(GenBank登录号:NM_176628)体外合成BoPAG19基因,PCR扩增目的基因,经双酶切后与真核表达载体pcMV3连接,构建pcMV3-BoPAG19重组质粒。采用Lipofectamine^®2000将重组质粒瞬时转染至HEK-293F细胞,SDS-PAGE法鉴定细胞培养上清中BoPAG19蛋白的表达,采用亲和层析法纯化BoPAG19蛋白。通过在线工具分析BoPAG19蛋白的疏水性、跨膜区域、信号肽、B细胞抗原、二级结构、三级结构和蛋白相互作用。【结果】成功构建pcMV3-BoPAG19重组载体,目的基因长约1 200 bp,表达蛋白约60 ku。生物信息学分析显示,BoPAG19蛋白编码380个氨基酸,其中含量最高的是丝氨酸(Ser),占比9.2%,含量最低的是色氨酸(Trp),占比1.6%;分子式为C₁₉₃₇H₃₀₂₈N₅₂₄O₅₃₂S₁₅,理论分子质量为41.8 ku,等电点为9.62,不稳定指数为40.75,在水中不稳定,脂肪系数为91.53,消光系数为52 370 mol^-1·cm^-1,具有水溶性;含有1个信号肽,无跨膜区域,有6个糖基化位点和11个B细胞表位;二级结构中α-螺旋、β-转角、无规则卷曲和延伸链占比分别为18.95%、6.32%、42.37%和32.37%,三级结构预测结果与二级结构一致。与BoPAG19互作的蛋白包括APLP2和APP,可能参与了妊娠期的神经调节。【结论】试验成功表达、纯化了BoPAG19蛋白,并分析了BoPAG19的生物信息学特征,为BoPAG19蛋白的结构和功能研究,以及BoPAG19诊断奠定基础。

Abstract: 【Objective】 The aim of this study was to amplify the bovine pregnancy-associated glycoprotein 19 (BoPAG19) gene,construct a eukaryotic expression vector,and detect its expression in HEK-293F cells.【Method】 BoPAG19 gene was synthesized in vitro according to the sequence of BoPAG19 gene (GenBank accession No.:NM_176628),and the target gene was amplified by PCR.After double enzyme digestion,the recombinant plasmid pcMV3-BoPAG19 was constructed and transfected into HEK-293F cells by Lipofectamine^® 2000.BoPAG19 protein was expressed in the supernatant of cell culture and determined by SDS-PAGE,and then was purified by affinity chromatography.The hydrophobicity,transmembrane region,signal peptide,B cell antigen,secondary structure,tertiary structure and protein interaction of BoPAG19 protein were analyzed using bioinformatics softwares.【Result】 The recombinant vector pcMV3-BoPAG19 was successfully constructed.The length of BoPAG19 gene was about 1 200 bp,and the expression protein was about 60 ku.Bioinformatics analysis showed that BoPAG19 protein encoded 380 amino acids,the highest content serine (Ser) was 9.2%,and the lowest content tryptophan (Trp) was 1.6%.The molecular formula of BoPAG19 protein was C₁₉₃₇H₃₀₂₈N₅₂₄O₅₃₂S₁₅,with theoretical molecular weight of 41.8 ku,isoelectric point of 9.62,instability index of 40.75,instability in water,fat coefficient of 91.53.The extinction coefficient was 52 370 mol^-1·cm^-1,and it was water-soluble.There was 1 signal peptide rigion and without transmembrane region.There were 6 potential glycosylation sites and 11 B cell epitopes.The secondary structure of BoPAG19 protein was composed of alpha helix (18.95%),beta turn (6.32%),random coil (42.37%) and extended chain (32.37%),respectively.The tertiary structure prediction result was consistent with the secondary structure.Proteins interacting results showed that BoPAG19 had interactions with APLP2 and APP,which might be involved in neuroregulation during pregnancy.【Conclusion】 In this study,BoPAG19 gene was successfully expressed and purified,and the bioinformatics characteristics of BoPAG19 were analyzed,which laid a foundation for the study of structure,function and diagnosis of BoPAG19 protein.

Key words: bovine pregnancy-associated glycoprotein 19 (BoPAG19); eukaryotic expression; bioinformatics

中图分类号:

董媛, 于鸿绪, 陈国良, 鲍东博, 崔嘉芮, 刘磊, 王会岩. 牛妊娠相关蛋白19真核表达载体的构建及生物信息学分析[J]. 中国畜牧兽医, 2022, 49(4): 1233-1243.

DONG Yuan, YU Hongxu, CHEN Guoliang, BAO Dongbo, CUI Jiarui, LIU Lei, WANG Huiyan. Construction of Eukaryotic Expression Vector of Bovine Pregnancy-associated Glycoprotein 19 and Its Bioinformatics Analysis[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(4): 1233-1243.

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