Objective   The bPAG16 gene related to bovine pregnancy was codon-optimized and synthesized to construct recombinant vector expressed in HEK293 cells for the development of a diagnostic tool on pregnancy of dairy cows.

  Methods  Bioinformatics were applied to codon-optimize and synthesize the bPAG16 gene followed by connecting it to the proEM vector by T4 DNA ligase. The resulting proEM-bPAG16 recombinant vector was transfected into HEK293 cells. The expressed gene was purified by Ni²⁺ affinity chromatography and verified by SDS-PAGE and western blotting.

  Result   With the optimization, the codon adaptation index (CAI) of the bPAG16 gene rose from 0.77 to 0.96 and the GC content from 48% to 58%. After enzymatic digestion, fragments of approximately 1 179 bp and 4 369 bp were obtained from the proEM-bPAG16 expression vector as expected. The nucleotide sequence of the inserted bPAG16 in the recombinant plasmid was same as that of the optimized gene and absent of any mutated amino acids. SDS-PAGE and western blotting showed that the 48kDa recombinant bPAG16 fusion protein was correctly expressed in the HEK293 cells with a purity greater than 90%.

  Conclusion   The successfully executed codon optimization and synthesis of bPAG16 gene provided the base material for further study on early diagnosis of pregnancy in dairy cows.