The Immune Effects And Mechanisms Of Opioid Peptides From Recombinant Pichia Pastoris

In recent twenty years, there are rapidly growing literatures described complexinterrelations of nerve, endocrine and immune, and the network of nerve-endocrine-immunewas advanced. Opioid peptides exert important modulatory role in nerve, endocrine andimmunity. The opioid peptides have extensive physiologic functions such as easing pain,relaxing, regulating immunity, improving appetite, regulating sleeping, restraining the motilityof gastrointestinal and the secretion of peptic and affecting procreation, regulatingcardiovascular function. They have potential availability in medicine, food and animalproduction industry.The objective of this paper was investigated on the optimum conditions for theexpression of recombinant opioid peptides in Pichia pastoris;new quantitative method ofopioid peptides;the non-specific, cellular and humoral immunity of three opioid peptidesobtained from separation and purification to mice in vitro and in vivo methods. It was studiedthe μ-,δ-opioid receptor and peptide transporters PepT1 gene expression in mice givenopioid peptides by intraperitoneal administration or drinking solution using the reversetranscription -polymerase chain reaction (RT-PCR) method. Finally, it was primarilyconducted that the opioid peptides exerted immunomodulatory effects on mice immuno-suppressed with cycolphophamide (Cy).1. Expression of opioid peptides in recombinant Pichia pastorisThe conditions for the expression of recombinant Pichia pastoris were optimized. Theconditions for the expression of recombinant Pichia pastoris were as follows: medium wasBMMY (pH 5.6), concentration of methanol was 0.75%, the initial concentration of cells was40 OD600, design temperature was 28℃, rotation speed was 250 rpm, the amount of peptidesexpressed with N-Tyr was 201.1mg/L.2. Studies on separation, purification and identifying activity of opioid peptidesA method was developed on the separation and purification of opioid peptides fromfermentation of recombinant Pichia pastoris. After fermentation of the recombinant Pichiapastoris, the supernatant was collected by centrifugation. The supernatant was ultrafiltratedand then treated by Sephadex G-10 chromatography. According to molecular weight of theopioid peptides using HPLC-MS, all possible opioid peptides in active component usingSephadex G-10 were ascertained. The active component was purified using the AKTA-100Purifier system employing DEAE-cellulose ion exchange chromatography and Sephasilpeptide C18 reverse phase column. Then six opioid peptides were obtained finally. The aminoacid compositions of six peptides were determined. The six opioid peptides showed obviouslyopioid activity. They were YGGFM,YGGFMT,YPFPGPI,YPFPGPIR,YPFPGPIRYG andYGGFMTSEK. Among of them, the YGGFMT , YPFPGPIR , YPFPGPIRYG andYGGFMTSEK were new opioid peptides.3. Immunomodulatory effects of the opioid peptides in vitroOP1 (YPFPGPI, β-CM-7), OP2 (YPFPGPIRYG) and OP3 (YGGFM, M-Enk) werechosen to study immunomodulatory effects. The effects of opioid peptides on mice werestudied using in vitro cell incubating. The results showed bidirectional regulation of the threeopioid peptides depending on the concentration. Moreover, the immunomodulatory effects ofOP1 on thymus and splenic cell might be attributed to interactions with the classical andnon-classical opioid receptors. The immunomodulatory effects of OP2 and OP3 on thymusand splenic cell might be attributed to interactions with the classical opioid receptors.4. The immunomodulatory effects of opioid peptides on mice in vivoThe immunodulatory effects of the opioid peptides were studied in mice. The differenteffects on immunity were found with three opioid peptides.(1) After the OP1(β-CM-7) was administrated by intraperitoneal injection or drinkingsolution, the proliferation of T and B lymphocyte induced with ConA or LPS and IL-1secretion of peritoneal macrophage of mice was significantly enhanced in vivo. At the sametime, it also significantly promoted IL-2 secretion of splenocytes and improved humoralimmunity of mice in intraperitoneal administration. When OP1 was administrated by drinkingto mice, spleen and thymus index of mice were greatly increased.(2) OP2(YPFPGPIRYG) suppressed significantly the proliferation of thymus and spleen Tlymphocyte induced with ConA and IL-2 secretion of splenocytes of mice, but significantlyenhanced nitric oxide secretion of peritoneal macrophage and improved humoral immunity ofmice (P>0.05) in intraperitoneal administration. When OP2 was given by drinking to mice,all testing immunity parameter had some change, but did not have statistical variation.(3) OP3 (M-Enk) significantly improved various immunity parameter of mice in vivo byintraperitoneal administration or drinking opioid peptides solution, involving the proliferationof thymus T cell, spleen T and B lymphocyte, nitric oxide and IL-1 secretion of peritonealmacrophage, IL-2 secretion of splenocytes and humoral immunity of mice.5.Primary research on immunomodulatory effects of opioid peptides in miceimmunosuppressed with immunosuppressive agent cycolphophamide (Cy).The immunomodulatory effects of opioid peptides on mice immunosuppressed bycycolphophamide (Cy) was primarily conducted. The results suggested that three opioidpeptides could withstand immunosuppressive of mice caused from Cy. The order of reversiblecapacity was OP3 (M-Enk)>OP1(β-CM-7)>OP2(YPFPGPIRYG).6. Effects of opioid receptor and peptide transporters gene expression of different tissuesin mice.After the mice administrated opioid peptides by intraperitoneal injection or drinking, theμ-,δ-opioid receptor and peptide transporters PepT1 gene expression were investigated bythe reverse transcription-polymerase chain reaction (RT-PCR) method. The results provideddirect evidence for tissue-specific opioid receptor gene expression in the immune system ofmice. The immunomodulatory effects of opioid peptides on thymus and splenic cell wereattributed to the interactions with the classical and non-classical opioid receptors. Theimmunomodulatory effects of OP1 and OP2 on mice were not attributed to the interactionwith the opioid receptors in central nerve system (CNS). OP3 might be interacted with the δ-opioid receptor in CNS to exert immunomodulatory effect. OP1, OP2 possibly interactedrespectively with theδ-opioid receptor and δ-and μ-opioid receptor in small intestine, butOP3 probably absorbed into blood then exert immunodulatory effects.