Molecular Characteristics,Isolation,Purification And Property Of Equine Chorionic Gonadotropin

Equine Chorionic Gonadotropin（e CG）,a reproductive hormone extracted from the serum of pregnant mares,has been listed in the veterinary pharmacopoeia,which can promote animal estrus,ovulation,induce twins and improve the quality of male semen.There are abundant horse resources in China,but the research on e CG is still shallow.The structure of e CG and how to perform its functions are still unclear.Moreover,the purification methods are still stagnated decades ago,and need to be improved and innovated urgently.In this study,we analyzed the structure of e CG by bioinformatics prediction,and predicted the structural region that determines the biological function of e CG according to its special biological function.We developed a new protein purification technology to isolate and purify e CG from pregnant horse serum with low specific activity and protein content,which laid a foundation for studying how e CG performs its biological function.The main research work is as follows:The total acidic amino acid residues（Asp+Glu）of e CGαand e CGβwere the same,but the total alkaline amino acid residues（Arg+Lys）of e CGαwere more than that of e CGβ.Both subunits were alkaline and uns Tablele.ECGαis hydrophilic,while e CGβis hydrophobic;e CGαand e CGβhave three strong hydrophobic regions and two strong hydrophilic regions;compared with e CGβ,d CGβand z CGβhave only 12 and 9 amino acid residues;the N-glycosylation sites of CGβin equine animals are similar,but the possibilities of O-glycosylation and their occurrence are different;The starting end ofα-amino group is mainlyα-helix,with stretching chain,β-rotation angle and irregular curl alternately arranged;the irregular curl of e CGβis the main structural element of the whole sequence;the three-dimensional structure of e CG is successfully docked,and the N-terminal of the complex is not tightly bound,while the C-terminal is folded to the protein core,and the hydrophilic core is wrapped in the middle of the whole protein.To develop a method for purifying e CG in laboratory,the best purification conditions were:adding 0.5mol/L partial phosphoric acid to p H3.5,0.1mol/L Na OH HPO₃ supernatant to p H 5,DEAE-FF chromatography using p H5.0 0.10mol/L Na Cl,acetic acid sodium acetate buffer elution,agarose gel chromatography using acetic acid sodium acetate buffer elution,collecting in the eluent,and making chromatography.Elution was carried out with p H 8.50.2mol/acetic acid-ammonium acetate buffer.The specific activity of e CG was 1490.33 IU/mg The specific activity of e CG measured by ovarian weight gain method in rats was1427.00+105.07 IU/mg and the recovery rate was 52.14%.Based on the above studies,a predictive structure region for the biological function of e CG was proposed,and a method for purification of e CG with low protein concentration and high specific activity was developed.It has certain guiding significance for the structure and function of e CG and the improvement of e CG purification system in China.