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

Abstract

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

CLC Number:

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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Construction of Eukaryotic Expression Vector of Bovine Pregnancy-associated Glycoprotein 19 and Its Bioinformatics Analysis

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