Preparation Of Polyclonal Anti-Sox2Antibody In Chicken And Its Preliminary Application

As the key gene to maintain the totipotency and self-renewal of embryonic stem cells, Sox2has been a research hotspot in recent years. The purpose of this study is to clone chicken Sox2gene, to obtain purified His-Sox2fusion protein by means of prokaryotic expression techniques, and finally to prepare polyclonal anti-Sox2antibody and evaluate its specificity and effectiveness.Total RNA was extracted from testis tissue of20day-old rooster, chicken Sox2cDNA was amplified by RT-PCR. The resulting PCR products were ligated to pMD18-T vector to construct recombinant plasmid pMD18-T-Sox2, which was submitted for DNA sequencing and bioinformatic analysis. Removed from pMD18-T-Sox2Sox2cDNA was subcloned to pET-30a to construct prokaryotic expression vector pET30a-Sox2. Confirmed by DNA sequencing, pET30a-Sox2was transformed into E.coli BL21(DE3) to express His-Sox2fusion protein under induction of IPTG, and the interest protein was detected by SDS-PAGE and Western blot. After optimization of induction condition, BL21cells, previously transformed by pET30a-Sox2, were cultured in large scale, then induced by1.Ommol/L IPTG at37℃for4h, and under denaturing condition, His-Sox2fusion protein was purified by using Ni-NTA agarose. With multiple subcutaneous injections, the purified His-Sox2protein was used as antigen to immunize New Zealand white rabbits for four times at an interval of2weeks. On the7th day after the last injection, blood was collected from the immunized rabitts, and serum, or anti-Sox2antibody, was isolated by centrifugation. Protein samples extricted from testis tissue of newly born rooster, as well as the purified His-Sox2fusion protein, was used as antigen to detect the specificity of anti-Sox2antibody by means of Western blotting assay. Meanwhire, testis paraffin sections were prepared, and the the specificity of anti-Sox2antibody was confirmed further by detecting the intrinsic Sox2expression in testis tissues by utilizing immunohistochemistry assay.The results showed that:(1) The open reading frame (ORF) of chiceken Sox2gene cloned here is composed of963nucleotide acids, encoding320amino acids, which respectively share95.8%and98.7%homology with another Gallus Sox2homolog (U12532.1) in nucleotide and amino acid sequences (2) SignalP3.0predicted the absence of signal peptide cleavage at the N-terminus of amino acids of chicken Sox2, indicating its whole ORF could be used to construct prokaryotic expression vector.(3) Recombinant pET-30a-Sox2plasmid expressed efficiently in E.coli BL21(DE3) under the optimal induction condition.(4) Western blotting assay proved that purified His-Sox2protein could specifically bind to the anti His-tag monoclonal antibody, and polyclonal anti-Sox2antibody was obtained from the immunizied New Zealand white rabbits (5) Western blotting assay also showed that the polyclonal anti-Sox2antibody could specifically bind to the intrisic Sox2protein in newly born rooster testis, as well as to the purified His-Sox2protein.(6) immunohistochemistry analysis convinced that the anti-Sox2antibody could also specifically react with intrsic Sox2protein in most of endothelial cells of seminiferous tubules and in minority stromal cells, which further proved the effectiveness and the specificity of the anti-Sox2antibody.In conclusion, chicken Sox2gene was cloned, the recombinant plasmid pET30a-Sox2was successfully constructed, His-Sox2protein was obtained, and finally high specific polyclonal anti-Sox2antibody in chicken was prepared. This study will lay a foundation for further research of the biological functions of chicken Sox2gene and the self-renewal mechanism of chicken pluripotent stem cells (e.g. iPS cells).