Cloning and expression of synthetic Mx1 gene involved in early maternal recognition pregnancy in cattle

Pregnancy-associated genes contribute to antiluteolytic mechanisms in cattle corpus luteum. Early luteolysis (days 12–14) was associated with differential expression of a number of genes in the corpus luteum, including Mx1 gene that was induced by interferon-tau (IFNT) as the main signal for maternal recognition of pregnancy in ruminants. This gene may be used as a gene target for the early detection method of cattle pregnancy. The objective of this study is to clone and express Mx1 gene in Escherichia coli systems to produce Mx1 antigenic protein that can be used to develop antibody polyclonal for early pregnancy detection. The 733 bp of Mx1 gene was obtained via artificial gene synthesis gBlock and was cloned into pJET 1.2/Blunt cloning vector. Then, Mx1 gene was constructed using the pET-32b expression vector in BamHI-HindIII restriction sites to generate the pET-Mx1 plasmid. The gene was fused with a nucleotides sequence encoding Trx-tag, His-tag, and S-tag producing ∼45 kDa of recombinant Mx1. Gene expression in the construct was controlled by the T7 promoter and Trx-tag start codon through IPTG induction. Results showed that E. coli carrying pET-Mx1 overexpressed a 45 kDa protein in induced culture as a soluble protein that was expressed in periplasm. Dot blot analysis using anti his ensures that E. coli expresses a recombinant Mx1 protein that can be used for the development of polyclonal antibodies.