Molecular cloning and expression of buffalo ESX-1 gene: A marker for X-chromosome bearing spermatozoa

Extra-embryonic tissue-spermatogenesis homeobox gene 1 (ESX-1), an X-linked gene that encodes homeobox protein, expresses specifically in X-chromosome bearing spermatozoa. The antibodies against this recombinant protein will be useful to assess the efficiency of sex sorted semen in buffalo. Therefore, the present study has been undertaken to clone and express the ESX-1 gene and then to study the characteristics of the expressed protein. In this investigation, the cDNA of ESX-1 was cloned, sequenced and expressed in E. coli. Predicted amino acid sequence of ESX-1 had shown highest homology with Bos taurus (91.6%). The homeobox region had shown the identity of 98.4%, 70.5%, 70.5%, 69.4% and 69.4% respectively with Bos taurus, Homo sapiens, Pan troglodytes, Macaca mulatta and Mus musculus. Expressed protein was purified using Cobalt-Agarose affinity column. SDS-PAGE showed a product ∼27kDa recombinant fusion protein. The purified product was confirmed by Western blot analysis using Ni-NTA anti hisitidine HRPase conjugate. The antibodies against ESX-1 protein can be used for evaluation of sex sorted semen.