Cloning of Gfrα1 Partial cDNA in Sheep and Prokaryotic Expression of Epitope Polypeptide

In order to clone the DNA sequence of Gfrα1 gene in sheep and deeply research the sheep spermatogonial stem cells(SSCs),in the present study,the partial sheep Gfrα1 cDNA were cloned using RT-PCR and molecular cloning technique.The cloned partial Gfrα1 gene was 1 312 bp in length,including an ORF of 1 311 bp encoding 437 amino acids.The nucleotide sequences of sheep Gfrα1 gene were compared with the counterpart sequences of Bos Taurus,homo sapiens,Pongo abelii,Rattus norvegicus and Mus musculus,and the nucleotide homology was 98.5%,91.3 %,91.0%,88.9% and 88.0%,respectively.According to the cloned Gfrα1 gene sequence,the epitope was forecasted and then the cDNA was inserted into pET-44a(+) vector.Subsequently,the recombinant was induced by IPTG.The recombinant epitope polypeptide was purified according to Affinity column chromatography.Western blot analysis indicated that the molecular weight of the expressed epitope polypeptide was the same as predicted size of approximate 18.2 ku.The data collected provided the important information for further making Gfrα1 gene polyclonal antibody and authenticating on molecular level for spermatogonial stem cells in sheep.