Bioinformatics analysis and polyclonal antibody preparation of Mesocricetus auratus DHX30

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2025-04-06 DOI:10.1016/j.pep.2025.106715

Keyi Wang , Jing Huang , Mingzhi Li , Tianxiang Han , Ju Yao , Decheng Kong , Bao Liu , Xiaojin Xia , Yunli Guo , Sha Li , Lingbao Kong

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引用次数: 0

Abstract

DHX30, as members of the DExD/H family, plays a crucial regulatory role in RNA metabolism and antiviral innate immune response. To facilitate comprehensive investigation of DHX30's multifunctional involvement in viral life cycles, a highly specific anti-DHX30 antibody is needed. In this paper, we used bioinformatic analyses combined with the Immune Epitope Database (IEDB) to identify immunodominant B-cell epitopes in the Mesocricetus auratus DHX30 protein. A hydrophilic peptide sequence demonstrating strong antigenic potential was selected for recombinant antigen production. The corresponding DHX30 truncated gene fragment was cloned into the pET-28a vector to generate the pET-28a-DHX30-N expression construct. Following transformation into E. coli BL21(DE3) competent cells, recombinant protein expression was induced and subsequently purified via Ni-NTA affinity chromatography. Six-week-old female KM mice were immunized intraperitoneally with the purified antigen. The antibody titer was determined by indirect ELISA, demonstrating high serum titers. Specificity validation through immunoblotting and immunocytochemical analyses confirmed the antibody's exceptional target recognition capability. These antibodies are expected to develop detection methods for further research.

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.