【作者】 韩飞；

【导师】 乐国伟； 施用晖；

【作者基本信息】 江南大学 ， 粮食、油脂与植物蛋白工程， 2005， 博士

【摘要】 阿片肽是一类生物活性多肽,在神经内分泌与免疫调节中起重要的调节作用,具有镇痛,镇静,调节免疫,促进摄食,调节睡眠,缓解压力和紧张,抑制胃肠道运动和消化液分泌,影响生殖和泌乳,调节心血管系统功能等生理活性,其在医药和动物生产中有着广阔的应用前景。本研究从基因重组阿片肽发酵表达条件的优化开始,使用新的阿片肽定量研究方法,进行了基因重组阿片肽的分离纯化工作,并结合国内外研究现状,着重研究了分离纯化得到的3种阿片肽对小鼠非特异性免疫,细胞免疫和体液免疫各项指标的影响;利用RT-PCR方法,从基因转录水平,研究了体内两种给药方式下3种阿片肽对小鼠体内不同脏器组织的μ阿片受体,δ阿片受体以及肽转运载体PepT1 mRNA丰度变化情况;初步研究了阿片肽对环磷酰胺诱导的免疫抑制小鼠的免疫调节作用。1.利用基因重组毕赤酵母发酵表达阿片肽对基因重组毕赤酵母的表达条件进行了优化。结果表明,毕赤酵母对外源阿片肽表达的最优摇瓶发酵条件为:培养基BMMY(pH5.6),甲醇诱导浓度0.75%,菌体起始浓度40 OD600,摇床设定温度28℃,转速250转/分钟。按以上条件进行表达,N-Tyr肽含量达到了201.1mg/L。2.阿片肽的提取分离和活性鉴定建立了一套基因重组阿片肽的分离纯化方法。将基因重组毕赤酵母经适宜的生长和表达培养后,所得的发酵液经离心得无细胞上清液,上清液超滤后过Sephadex G-10柱,将所得具有阿片活性的粗品用HPLC-MS分析,依据分子量确定活性组分中可能存在的阿片肽,然后根据这些阿片肽的等电点和疏水性,利用?KTA Purifier 100快速纯化系统的DEAE-阴离子交换柱和Sephasil peptide C18反相高压液相柱分离得到六种阿片肽,鉴定纯度后测定其氨基酸组成。六种阿片肽分别为YGGFM、YGGFMT、YPFPGPI、YPFPGPIR、YPFPGPIRYG和YGGFMTSEK,其中YGGFMT、YPFPGPIR、YPFPGPIRYG和YGGFMTSEK目前尚未见报道。3.不同类型阿片肽对小鼠免疫细胞的免疫作用从分离纯化出的六种阿片肽中选出OP1(YPFPGPI,β-CM-7)、OP2(YPFPGPIRYG)和OP3(YGGFM,M-Enk)三种阿片肽进行后续的研究工作。利用体外细胞培养法研究三种阿片肽对小鼠的免疫作用。研究发现,三种阿片肽对体外培养小鼠的免疫细胞呈双向调节作用。阿片肽的浓度一般在10-7mol/L时,刺激或抑制作用表现最强。且体外培养实验表明,OP1对胸腺和脾细胞的作用是通过胸腺和脾细胞上的经典阿片受体和非经典阿片受体(能够被阿片受体拮抗剂纳酪酮逆转的为经典阿片受体,反之为非经典阿片受体)共同完成的;OP2和OP3对胸腺和脾细胞的作用是完全通过胸腺和脾细胞上的经典阿片受体完成的。4.体内法研究不同类型阿片肽对小鼠的免疫作用采用腹腔注射和饮水中添加阿片肽的方法研究阿片肽对小鼠的免疫调节作用。研究更多还原

【Abstract】 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.更多还原