Screening Porcine Growth Hormone Analogue Peptide And Identification Of Its Activity

Porcine growth hormone(pGH)is produced and secreted by the pituitary gland,191 amino acids of polypeptide hormones,Porcine growth hormone has diverse biological rol es,to promote the decomposition of fat,bone cell proliferation,and accelerate metabolis m.Since people found that porcine growth hormone have an important role.Scientists tr y to extract the porcine growth hormone from pituitary tissue,but due to growth hormo ne secretion by periodicity,less secretion,complex technique for purification,until now,the study on growth hormone research is only in lab level,not apply for large-scale live stock production.With the development of science and technology,in late 80,along wit h the innovation of molecular biology technology,Scientists produced recombinant growt h hormone by way of gene expression,which provide the necessary conditions for using of porcine growth hormone in animal production,scientists began to apply for porcine growth hormone in small scale,and received good effects.Because of hormone residues in animal products,our country and most countries of the world prohibited use of exo genous growth hormone in animal production.Therefore,study on growth hormone in-de pth and promoting growth mechanism and looking for safe and effective alternatives for GH to animal growth has been an important research direction.It mainly includes the f ollowing aspects:(1)the preparation of antibody(IgG)against growth hormone receptor can mimic the role of growth hormone,this is based on the ligand that induced recept or dimerization.(2)by growth hormone molecules on a section of activity sequence(mai nlygrowth hormone and receptor binding sites)simulation of actions of growth hormone s(3)based on the Jerne's the immune network theory,by anti-idiotypic antibody strategy,namely the growth hormone" image " of anti-idiotypic antibody to mimic growth horm one.(4)by the inhibition of somatostatin role,to promote the secretion of growth hormo ne.This study was sopported by the National Natural Science Fund(number:30871842),m ainly in the following several aspects of research.(1)The monoclonal antibodies against porcine growth hormone had been prepared in our lab and obtained the national invention patent(patent number:CN 1296385C).phage display random 12 peptide library was used to screen amino acid sequence by using ant ibodies as target,by means of "adsorption,elution,and amplification" after panning for t hree rounds,screened phage which specifically bound to monoclonal antibody against po rcine growth hormone.Then its ssDNA was extracted and sequenced,and the resulting peptide sequence were analyzed,were screened with the same amino acid and the amino acid sequences of high frequency,then by analysis of amino acids sequence,and results showed 2 groups of higher homologous amino acid sequences,and combination with co mputational biology analysis,Porcine growth hormone analogue peptide,RPCLPHCNSTS D were obtained.(relevant research has been declared a national invention patent,the nu mber for patent applications is 201110442548.8).(2)liver cell model was established in vitro,the activity of porcine growth hormone analog peptide activity was tested by confocal microscopy,flow cytometry analysis,com petitive binding receptor analysis techniques to verify the porcine growth hormone mimet ic peptide with liver cell surface growth hormone receptor combination.Western-blot and flow cytometry confirmed that the mimetic peptide of porcine growth hormone can activ ate growth hormone receptor in liver cells and trigger the JAK-STAT signal transduction pathway,and porcine growth hormone mimetic peptide can promote hepatic IGF-I releas e.(3)the results of the signal transduction induced by porcine growth hormone mimeti c peptide indicated that the peptide and porcine growth hormone activate the same signa 1 transduction pathway.In addition,porcine growth hormone mimetic peptide activate the signal transduction by a dose-dependent manner,and showed the bell-shaped activation c urve(the largest concentration was 1/2KD value).This indicates that the porcine growth h ormone mimetic peptide may activate the growth hormone receptor probaly by mode of dimerization.(4)We initial preliminary exploration from negative regulation.I constructed swine s omatostatin receptor model,and by the computer software and the server evaluates the s omatostatin receptor model,the results show that it is correct,this also provide certain t heoretical foundation on mechanism for studies of growth hormone regulation.In summary,porcine growth hormone mimetic peptide had been obtained by the use of phage display technology,we obtain the core amino acid sequence of porcine growth hormone mimetic peptide.To confirm its activity,the liver cells were used as a model,porcine growth hormone mimetic peptide indicate a specific combination with porcine growth hormone receptors expressed on the porcine liver cells,trigger the JAK-STAT signal transduction pathway,and stimulate IGF-I release in liver cells.In addition,We initial preliminary exploration from the porcine growth hormone negative feedback regulation mechanisms.In present study,we provide a theoretical basis for the regulation of promotion of animal growth,and explore the mechanism of promoting growth by immune regulation,and develop alternative new safe and effective growth hormone active peptide vaccine,all of these lay the theoretical and technical basis.