Pregnancy-associated glycoproteins (PAGs) are widely used as powerful markers for early pregnancy diagnosis in livestock. To improve expression efficiency of recombinant ovine pregnancy-associated glycoprotein-7 (ovPAG-7) in HEK293 cells, the codon usage bias of the ovPAG-7 gene was analyzed using bioinformatic approaches, after which the DNA sequence encoding ovPAG-7 was designed, synthesized, and expressed in HEK293. The structure and function of ovPAG-7 were predicted using bioinformatics software and online databases. The results showed that the effective number of codons (NEC) of the ovPAG-7 gene was 56.82, indicating that the ovPAG-7 gene was weakly biased. ovPAG-7 gene had 26 biased codons (relative synonymous codon usage (RSCU) > 1), 15 of which were biased towards G/C at the third position. After codon optimization, the codon adaptation index of the ovPAG-7 gene increased from 0.74 to 0.96, and its GC content changed from 46.6 to 58.6%. The amino acid sequence encoded by the optimized gene was entirely consistent with those published in Gen Bank. Western blot analysis indicated that the recombinant ovPAG-7 protein with a relative molecular mass of 48 kDa was successfully expressed in HEK293 cells. The bioinformatics prediction results showed that ovPAG-7 protein contained 3 N-glycosylation sites, 13 B-cell epitopes, and a signal peptide consisting of 15 amino acids at the N terminus. The secondary structure of the ovPAG-7 protein was predicted to consist of random coils (46.85%), extended strands (32.05%), α-helices (16.16%), and β-turns (4.93%). This study provided a tool for the screening of monoclonal antibodies and functional research on ovPAG-7.
Keywords: Codon optimization; Codon usage bias; Eukaryotic expression; Pregnancy–associated glycoprotein; Recombinant proteins.
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