Cloning And Prokaryotic Expression Of The Inhibin-α-Subunit Maturate Region Coding Sequence In Northeast White And Zi Goose

Inhibins are a hormone of biological activity to suppress the synthesis and releasing of FSH from pituitary, and so ovulation and egg laying are suppressed in poultry. The emphasis of this study focused on cloning and prokaryotic expression of mature inhibinα-subunit cDNA of Northeast white and Zi goose in BL21(DE3) strain. The aim of the present study is to provide theoretical foundations for the effect of recombinant protein on poultry reproduction.The Inhibinα-subunit coding sequence of Northeast white and Zi goose optimized according to common codons of E. coli. The ends of F1 and R1 were designed with recognition sites restriction enzyme, Sac I and Xho I. Maturation zone of Inhibin-αsubunit was amplificated from granular cell of Northeast white and Zi goose ovarian follicle by RT-PCR. The recombinant plasmid pMD-18T-INH-αwas transformed by inserting the fragment of 354 bp obtained by PCR into pMD18-T Simple vector. The positive clone from XL1-Blue sensitive cell was evaluated by double enzyme digestion and PCR. DNA sequence analysis showed that the cloned sequence was the very fragment we designed. Those showed that we obtained clone vector of Northeast white and Zi goose inhibin-αsubunit. Compared to the chicken 's maturation region inhibin-αsubunit reported in GenBank, the homology rate in nucleotide sequence of Northeast white and Zi goose are 94.44% and 94.74%, and that of amino acid sequence are 96.5% and 97.35%, which indicates that the inhibin-αsubuntit is highly conserved among different poultry species.This extracted plasmid was digested with Sac I and Xho I and ligated the pET-32a vector digested with the same enzymes using T4 DNA ligase. Positive plasmids were generated by transforming the plasmid INH-pET-32a to the competent cell of BL21(DE3).The positive bacterium was induced by IPTG. The expression of inhibin-αsubunit was observed on Tricine-SDS-PAGE. Mus musculus albus were immunized with inhibin-αsubunit fusion protein and monomer protein by thrombin digested. Characteristic protein band of prepared antibody that inhibin-αsubunit appeared clear by Western blotting,, the result confirmed that fusion protein and monomer protein by thrombin digested of inhibin-αsubunit has favourable immunogenicity.