豬生長激素化學修飾之研究

Abstract

遺傳工程生產之豬生長激素(Recombinant Porcine Growth Hormone, rPGH)，使用2-Nitrophenyl-sulfenyl chloride (NPS-C1)與Glutaraldehyde (GA)做化學修飾。由Methanesulfonic acid水解分析與spectrophotometry法，證明 NPS－C1作用在rPGH之Tryptophan上，且其反應程度接近完全。以放射免疫測定法(Radioimmunoassay)測定，其結合能力降至未經修飾的rPGH之1/10。以腦下腺切除之Long Evans雌鼠做生物檢定(Bioassay)，初步證實在高劑量下，NPS-rPGH和rPGH具同等的生物活性(Biological activity)。 Glutaraldehyde可使豬生長激素產生分子間和分子內之架橋反應(Cross-linking)。在大於0.1％的Glutar－aldehyde作用下，其產生之單元體(Monomer)與雙元體(Dimer)在SDS－PAGE上之移動速率較未反應者快。在0.5% Glutaraldehyde溶液中反應後之GA-rPGH以HPLC之Gel filtration Column (Waters protein PAK 300sw)分離得到其單元體(M-GA-rPGH)與雙元體(D－GA－rPGH)。以放射免疫測定法比較M－GA－rPGH、D－GA－rPGH、rPGH和抗體結合之競爭能力，結果經修飾過之rPGH，其競爭能力降低至1/3-1/5。Glutaraldehyde濃度對反應程度影響極大。 將鴨生長激素(duck Growth Hormone, dGH)之互補基因(cDNA)構建於pSV-neo之SV40 promoter後面，感染( Transfect)到Cos-I細胞後，其培養基液經放射免疫測定，初步證實具瞬間表現(Transient expression)之現象。

2-Nitrophenylsulfenyl chloride (NPS-C1) reacted with the tryptophan residue of rPGH. The product was then purified through G-25 column which was pre-equilibrated and eluted with 0.1M NH4OAc pH4.5. The radioimmunoassay proved that the purified NPS-rPGH retained 1/10 binding activity with antibody (monkey antiserum against rPGH) in comparison with original rPGH. This phenomenon implied the conformation change of the NPS-rPGH. Also,the bioassay (Tibia assay) proved the NPS-rPGH retained full growth-promoting activity. There are intramolecular and intermolecular cross-linking reactions between rPGH and high concentration of glutaraldehyde. Monomer (M-GA-rPGH) and dimer (D-GA-rPGH) were purified by using the HPLC gel filtration column.The mobility of the monomer was faster than the original rPGH on SDS PAGE. It implied that the formation of intramolecular lysine cross-linking would provides a more stable conformaton on SOS PAGE. The dimer, trimer, tetramer and oligomer products proved the reaction of intermolecular cross-linking. Comparing to original rPGH, the binding activity of M-GA-rPGH and D-GA-rPGH with antibody reduced to 1/3-1/5 by radioimmunoassay.