Construction of Bovine CypA Gene Expression Vector and Validation of Its Expression in CHO-K1 Cells

TL;DR

Researchers created a system to produce bovine cyclophilin A in lab cells, which could help study and treat mastitis in cows.

Contribution

A novel eukaryotic expression vector for bovine cyclophilin A was constructed and validated in CHO-K1 cells.

Findings

A eukaryotic expression vector containing the bovine CypA gene was successfully constructed and confirmed via sequencing.

CHO-K1 cells transfected with the vector showed stable EGFP fluorescence and CypA protein expression confirmed by Western blot.

The system provides a reliable source of recombinant CypA for future functional and therapeutic studies.

Abstract

Bovine mastitis is a major disease affecting dairy cow health and milk production, and current treatment options still have important limitations. Previous studies have shown that a protein called cyclophilin A is closely associated with inflammatory responses during mastitis, but further research requires a stable and reliable source of this protein. In this study, we established a method for the stable expression of bovine cyclophilin A in mammalian cells and demonstrated that these cells can continuously express the target protein. This work provides a solid technical foundation for future studies on the role of this protein in bovine mastitis and for the development of related diagnostic or intervention approaches, and it may contribute to improving the prevention and control of bovine mastitis.

Bovine mastitis remains a globally prevalent disease, with the limitations of antibiotic-based treatments—such as the rise in antimicrobial resistance and the presence of drug residues—highlighting the urgent need for alternative therapeutic approaches. Inflammation is intricately linked to various cytokines and immunomodulatory proteins, among which cyclophilin A (CypA) serves as a pivotal inflammatory mediator, significantly contributing to the initiation and amplification of inflammatory responses under such conditions. The acquisition of high-purity recombinant protein is a fundamental prerequisite for in vitro functional studies of bovine CypA. This study aimed to construct a eukaryotic expression vector for bovine CypA and verify its expression in CHO-K1 cells. Utilizing the bovine CypA gene sequence available in GenBank, the coding region was artificially synthesized and optimized for codon usage, subsequently being inserted into the pPB[Exp] backbone vector via BsrGI and BstEII double digestion. The resulting polycistronic expression vector contained a CAG promoter driving the CypA transcription, an EF1α promoter driving the EGFP reporter gene, a PGK promoter controlling the puromycin resistance gene, and a C-terminal His-tag. Restriction enzyme digestion and bidirectional Sanger sequencing confirmed that the inserted fragment sequence was completely consistent with the optimized design. Robust EGFP fluorescence was observed 24 h post-transfection and remained stable after puromycin selection. qPCR analysis showed that the Ct value of CypA in the experimental group was 16.20 ± 0.04, while no amplification signal was detected in the control group. Additionally, Western blot analysis identified a CypA-specific band at approximately 18 kDa, confirming the correct expression of the exogenous CypA protein in CHO-K1 cells. Collectively, these results demonstrate the successful construction and validation of a bovine CypA eukaryotic expression vector. The established CHO-K1 expression system exhibited stable and efficient expression, thereby providing a robust foundation for future research on the production and application of recombinant CypA protein.

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