Molecular Cloning And Prokaryotic Expression Of The Leghorn Chicken FOXL2 Gene

The study on sex determination and sex differentiation has a great significance to poultry production.FOXL2 is a single exon transcription factor located in autosome.It is the first discovered mammalian ovarian differentiation marker and plays an important role in ovarian development and maintenance.To obtain the reorganization Leghorn protein FOXL2 with biological activity by genetic engineering techniques has great significances to study the effect of FOXL2 on the sex differentiation and gonadal development in the chicken.Two pairs of primers?P1-P2 and ePl-eP2?were designed,based on the published nucleotide sequence of the Gallus FOXL2 gene?GenBank accession:NM₀01012612.1?.The full-length coding sequence of the Leghorn FOXL2 DNA was amplified by PCR using a template of the SPF Leghorn whole blood.The product was cloned into the pMD18-T vector.Thus we got the recombinant cloning vector pMD18-T-FOXL2.Sequence analysis showed that cloned target sequences contained the Leghorn FOXL2 gene,which meant that we successfully constructed the pMD18-T-FOXL2 plasmid.The Leghorn FOXL2 sequence was analyzed and predicted by bioinformatic tools.The results showed that Leghorn FOXL2 gene was 1130 bp?GenBank accession:FJ708868?in length with a 918 bp open reading frame?ORF?and encoded 305 amino acids.The nucleotide sequence of CDS shared 99.8%,80.0%,80.4%,80.3%,80.5%,81.5%,77.7%,71.8%and 75.5%homology with the FOXL2 mRNA of Gallus,Homo.sapiens,Bos.Taurus,Sus.scrofa,Mus.musculus,Oryctolagus cuniculus,Xenopus laevis,Danio rerio and Oreochromis niloticulus respectively,and the homology of deduced amino acids were 99.7%,64.7%,64.7%,65.4%,63.5%,63.8%,79.7%,78.3%and 79.9%respectively.It was predicted that FOXL2 mainly existed in the nucleus,which contained one conserved domain,but no signal peptide,transmembrane region and splice sites.It contained two protein kinase C phosphorylation sites,one cAMP and cGMP dependent protein kinase phosphorylation site,two N-glycosylation sites,four N-myristoylation sites and three casein kinase ? phosphorylation sites.The best antigen epitope probably located in or was adjacent to the regions of 37-49?KGPEKPDPSQKPP?,82-91?PFYEKNKKGW?and 133-147?KGNYRRRRRMKRPFR?.Using pMD18-T-FOXL2 as the template,FOXL2 gene was amplified by PCR with restriction sites,and then was directionally cloned into the pET28a?+?vector.The recombinant expression plasmid in the E.coli BL21?DE3?was induced by 0.5 mmol/L IPTG.SDS-PAGE analysis showed that the induced expressed protein was about 37 ku.Western blot revealed that the expressed protein was the His tag fusion protein,which indicated that the recombinant prokaryotic expression vector expressed the fusion protein successfully.The cloning and expression of the fusion protein was successful.The cloning and expression of the Leghorn FOXL2 gene established the foundation for further study on its biological function.