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

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.