固相合成胸腺五肽的分离纯化与鉴定

目的 建立胸腺五肽分离纯化、分析鉴定方法,有效提高胸腺五肽含量。方法 将标准Fmoc方法固相合成的胸腺五肽以Sephadex G-25凝胶柱使粗肽脱盐,以循环制备液相色谱仪进行粗肽纯化,以质谱法进行分子质量的鉴定。结果 该法得到胸腺五肽的纯度为99.02% ,分子质量测定值与理论值相符。结论 建立了高效,简便的胸腺五肽分离纯化方法, 为工业化生产提供了实验依据。     

海胆黄多糖的分离、纯化及免疫活性测定

目的从光棘球海胆中分离纯化海胆黄多糖(polysaccharidefromtheeggsofStrongylocentrotusnu-dus,简称SEP),确定其纯度和分子量,并现察其免疫活性。方法海胆黄先经丙酮脱脂,根据正交实验和活性分析确定最佳热水提取条件,然后热水提取、去蛋白、醇沉得海胆黄粗多糖。粗多糖经超滤、DEAESepharoseFastFlow及SephacrylS-400柱层析纯化得多糖精品SEP。经高效液相色谱、聚丙烯酰胺凝胶电泳及纸层析鉴定其纯度。高效凝胶渗透色谱法(HPGPC)测定其分子量。体外脾淋巴细胞增殖实验测定其免疫活性。结果从海胆黄中分离纯化得到的均一多糖组分SEP,经检测其分子量为1950KD左右。脾淋巴细胞增殖实验表明SEP可显著促进脾淋巴细胞的增殖。结论从海胆黄中分离纯化得到的均一多糖组分SEP具有较强的体外免疫活性。     

胸腺五肽对慢性支气管炎患者血清T细胞亚群水平的影响

老年免疫功能减退是导致衰老及多种老年病的重要原因,免疫功能减退主要表现在细胞免疫方面,尤其是T细胞的变化,因此,T淋巴细胞亚群测定可以作为免疫功能检测指标之一。本研究通过对老年慢性支气管炎患者使用胸腺五肽治疗前后T淋巴细胞亚群水平的变化,以探讨胸腺五肽刘老年人免疫功能的影响。     

芍芪多苷对迟发型变态反应小鼠细胞免疫功能的影响

目的探讨在不同免疫状态下,芍芪多苷(Shaoqid-uogan,SQDG)对迟发型变态反应(DTH)小鼠细胞免疫功能的作用。方法采用2,4-二硝基氯苯(DNCB)诱导小鼠DTH模型及环磷酰胺(Cy)诱导小鼠免疫低下或亢进模型。SQDG予小鼠连续灌胃7d。MTT法检测ConA诱导的小鼠胸腺T淋巴细胞增殖,小鼠胸腺细胞增殖法检测ConA诱导的小鼠胸腺T淋巴细胞培养上清中白细胞介素2(IL-2)的活性。结果在小鼠DTH模型,SQDG(120mg·kg-1)可降低耳肿胀、胸腺指数、ConA诱导的胸腺T淋巴细胞增殖反应以及ConA诱导的胸腺T淋巴细胞培养上清中IL-2的活性。DNCB初次致敏当日腹腔注射(ip)Cy(150mg·kg-1)可以造成DTH低下模型,致敏前3 d ip Cy(250mg·kg-1)可造成DTH亢进模型;SQDG(60和120mg·kg-1)能明显上调DTH低下模型小鼠耳肿胀、ConA诱导的小鼠胸腺T淋巴细胞增殖及小鼠胸腺T淋巴细胞培养上清中IL-2的水平;且能明显下调DTH亢进模型小鼠上述指标的水平。结论SQDG对小鼠细胞免疫具有双向调节的作用。     

海绵Axinella verrucosa中溴吡咯生物碱的研究

目的分离鉴定海绵Axinella verrucosa中的溴吡咯生物碱,并进行体外免疫活性评价。方法利用减压柱层析、Sephdex LH-20凝胶柱层析、半制备HPLC等色谱方法分离纯化,运用NMR、MS等波谱方法并结合文献鉴定化合物结构,采用体外脾淋巴细胞增殖实验评价化合物的免疫活性。结果分离得到5个溴吡咯生物碱,分别鉴定为hymenidin(1)、stevensine(2)、4-bromopyrrole-2-carboxylic acid(3)、4,5-dibromopyrrole-2-carboxylic acid(4)和3,5-dibromo-2-pyrrolecarboxylic acid(5)。化合物1能够增强ConA诱导的T淋巴细胞增殖,化合物2能够增强LPS诱导的B淋巴细胞增殖。结论化合物4为首次从该种中分到,5为首次从该属中鉴定。化合物1和2具有一定的增强免疫作用。     

兔、牛胸腺肽的制备及其生化性质的研究

<正> 目前已公认胸腺为免疫系统的主宰腺体,胸脉肽是胸腺分泌的一组多肽类激素,其作用在体内外可促进淋巴细胞合成DNA,诱导T细胞获得表面抗原,细胞免疫重建。临床上已用于纠正各种免疫功能紊乱性疾病。自从Goldstein等于1966年首先从小牛胸腺提取并命名为胸腺素以来,许多学者对胸腺提取物的研究逐渐深入,相继从猪、人胚等胸腺中分离得到类似物,兔胸腺肽的研究尚未见报道。     

海藻硫酸多糖对小鼠免疫功能的调节作用

目的：观察海藻硫酸多糖（SPS）对正常小鼠和免疫低下小鼠免疫功能的调节作用。方法：测定小鼠腹腔巨噬细胞吞噬鸡红细胞的能力、碳粒廓清能力、血清溶血素形成,,并进行二硝基氟苯诱导小鼠迟发性变态反应实验、T,B淋巴细胞增殖实验,考察SPS对正常小鼠免疫功能的影响;测定环磷酰胺致免疫低下小鼠的血象、胸膜指数和T淋巴细胞亚群,进行T,B淋巴细胞增殖实验,考察SPS对免疫低下小鼠免疫功能的影响。结果：SPS能增强正常小鼠腹腔巨噬细胞吞噬鸡红细胞的能力,高剂量SPS能增强小鼠碳粒廓清能力,高、中剂量SPS能提高小鼠血细胞凝集程度,SPS体外对T,B淋巴细胞有促增殖作用。对于环磷酰胺所致免疫低下小鼠,SPS可以通过调节血象、增加胸腺指数、提高T淋巴细胞和CD8^ 细胞数目、促进T,B淋巴细胞增殖来缓解小鼠免疫功能的低下。结论：SPS是一种免疫调节剂,具有免疫调节和抗病毒作用,是新一代抗HIV药物的发展方向。     

生津润燥颗粒对干燥综合征模型小鼠的影响

目的:研究生津润燥颗粒对干燥综合征模型小鼠的影响。方法:利用免疫诱导法建立干燥综合征小鼠模型,考察生津润燥颗粒对模型小鼠颌下腺组织形态学变化、颌下腺与胸腺脏器指数及唾液分泌量的影响,同时观察小鼠血液T淋巴细胞阳性率及脾脏T淋巴细胞、B淋巴细胞增殖能力。结果:生津润燥颗粒能促进干燥综合征模型小鼠颌下腺组织修复,增加颌下腺指数,降低胸腺指数,增加唾液分泌量,并使血液T淋巴细胞阳性率及脾脏T淋巴细胞、B淋巴细胞增殖转化能力降低。结论:生津润燥颗粒对干燥综合征模型小鼠具有一定的治疗作用。     

枸杞粗多糖的免疫活性

为进一步分离纯化枸杞多糖并进行深入的免疫药理学研究 ,应用淋巴细胞增殖 ,溶血空斑 ,杀伤性T淋巴细胞 (CTL)杀伤活性测定 ,酶联免疫测定法和噻唑蓝等方法研究了枸杞粗多糖 (LBP)的免疫活性特点 .结果发现 :口服给予LBP对LACA小鼠脾细胞增殖反应和抗体生成反应均具有明显的促进作用 ,但对CTL细胞特异性杀伤P815瘤细胞的杀伤活性无明显影响 ;明显增加快速老化模型小鼠(SAMP8)脾抗体生成细胞的数目 ,升高脾细胞产生抗体IgG水平 .结果提示 ,LBP是枸杞子中主要免疫活性成分 ,促进或改善体液免疫功能可能是其对免疫系统的主要作用之一 .     

小牛胸腺肽类提取物(T.P)的制备及其对小鼠腹腔巨噬细胞吞噬功能的影响初步报告

胸腺是产生免疫的组织器官之一。Goldstei-n、A、L和WhiteA 1966’从小牛胸腺中分离出一种激素,称胸腺素(Thymosin)、还有人从胸腺中提取出称为THE激素和TF激素等,均可将淋巴母细胞转化为具有活性的淋巴细胞,促进T淋巴细胞的分化。罗马尼亚布加勒斯特内分泌研究所     

精子抗原肽P10G和YLP12的合成及在ELISA检测中的意义

目的 人工合成精子肽P10G和YLP12,以此为抗原建立检测抗精子抗体(AsAb)的酶联免疫吸附测定法(ELISA).方法 用PS3全自动合成仪合成精子肽P10G和YLP12,经反相高效液相色谱纯化和质谱鉴定.分别包板制备ELISA试剂盒,用以检测血清中的AsAb.结果 成功合成两种目的肽,质谱结果显示其相对分子质量均与理论相对分子质量相吻合,高效液谱法(HPLC)结果显示其纯度为97.5％和97.8％.以该两种合成肽为抗原,建立AsAb的ELISA,与商品化试剂盒比较均显示极好的一致性(Kappa值为0.77和0.79,＞0.75).结论 全自动固相多肽合成技术可用于合成高纯度的精子抗原肽,所合成的P10G和YLP12均具有较好的抗原活性,可作为包被抗原用于AsAb的ELISA检测.     

宫颈癌治疗性多肽疫苗的合成及其诱导细胞毒性T淋巴细胞效应的研究

目的：设计并合成宫颈癌治疗性多肽疫苗，并对其免疫学效应进行研究。方法：以芴甲氧羰基固相法合成多肽，用高效液相色谱分析多肽的纯度，对所合成的多肽采用质谱进行鉴定，用标准^51Cr释放试验检测各肽在HLA—A2转基因鼠中诱导的表位特异性细胞毒性T淋巴细胞活性。结果：合成的多肽经纯化后纯度分别为86．5％、81．8％和98，5％，质谱分析各肽的相对分子质量分别为3121、2580和815，与理论值相符。其在HLA—A2转基因鼠中可诱导出强的表位特异性细胞毒性T淋巴细胞活性。结论：合成的多肽为目标肽，将辅助性T细胞表位及脂类分子构建于分子内部可提高细胞毒性T淋巴细胞表位肽的免疫原性。     

Systematic study of the substituted active C-terminus of hirudin

Abstract: Hirudin, a potent clinical thrombin inhibitor from Hirudo medicinalis, consists of 65 amino acids in a single chain. In this paper, we systematically synthesize a series of C-terminal (desulfo hirudin^45–65) peptides substituted by 20 natural l -amino acids via the Multipin method. The resulting peptide library is subsequently screened using an α-thrombin-mediated fibrinogen clotting assay and α-thrombin-induced amidolytic hydrolysis assay.
     

以精子抗原肽为抗原的抗精子抗体ELISA检测方法的建立

目的建立组合多个精子抗原肽为抗原的检测抗精子抗体的酶联免疫吸附测定(ELISA)法。方法用PS3全自动多肽合成仪合成4个特异精子肽,并经反相高效液相色谱(RP-HPLC)纯化和质谱鉴定。用ELISA检测4个合成肽的抗原性,基于对这些合成肽抗原活性的评价,将4个精子抗原肽混合后(组合肽)作为抗精子抗体(AsAb)检测用抗原。结果合成了实验所需要的抗原性肽段,RP-HPLC结果显示纯度达95%以上。以组合肽为抗原建立的检测AsAb的间接ELISA方法,与商品化试剂盒相比符合率为95.6%。结论固相多肽合成技术可用于合成高纯度的精子抗原肽,以组合的精子抗原肽作为包被抗原建立的ELISA方法可提高AsAb检测的灵敏度。     

固相法合成心肌兴奋肽及其类似物

用Boc-和Tos-基团分别保护氨基和侧链胍基,以1%交联度聚苯乙烯二苯甲氨基树脂为载体,用DCC固相法合成肽,HF断裂肽树脂键和去除侧链保护基团,粗产物经高效液相层析纯化,合成了心肌兴奋肽Phe-Met-Arg-Phe-NH_2及其类似物Phe-Pro-Arg-Phe-NH_2,并观察了此二种肽对大鼠血压和心率的影响。     

Facile solid‐phase synthesis of C‐terminal peptide aldehydes and hydroxamates from a common Backbone Amide‐Linked (BAL) intermediate*†

Abstract: C‐Terminal peptide aldehydes and hydroxamates comprise two separate classes of effective inhibitors of a number of serine, aspartate, cysteine, and metalloproteases. Presented here is a method for preparation of both classes of peptide derivatives from the same resin‐bound Weinreb amide precursor. Thus, 5‐[(2 or 4)‐formyl‐3,5‐dimethoxyphenoxy]butyramido‐polyethylene glycol‐polystyrene (BAL‐PEG‐PS) was treated with methoxylamine hydrochloride in the presence of sodium cyanoborohydride to provide a resin‐bound methoxylamine, which was efficiently acylated by different Fmoc‐amino acids upon bromo‐tris‐pyrrolidone‐phosphonium hexafluorophosphate (PyBrOP) activation. Solid‐phase chain elongation gave backbone amide‐linked (BAL) peptide Weinreb amides, which were cleaved either by trifluoroacetic acid (TFA) in the presence of scavengers to provide the corresponding peptide hydroxamates, or by lithium aluminum hydride in tetrahydrofuran (THF) to provide the corresponding C‐terminal peptide aldehydes. With several model sequences, peptide hydroxamates were obtained in crude yields of 68–83% and initial purities of at least 85%, whereas peptide aldehydes were obtained in crude yields of 16–53% and initial purities in the range of 30–40%. Under the LiAlH₄ cleavage conditions used, those model peptides containing t‐Bu‐protected aspartate residues underwent partial side chain reduction to the corresponding homoserine‐containing peptides. Similar results were obtained when working with high‐load aminomethyl‐polystyrene, suggesting that this chemistry will be generally applicable to a range of supporting materials.     

Thymosin-like peptides as potential immunostimulants. Synthesis via the polymeric-reagent method.

This paper reports our attempt at designing new immunostimulating peptides which are chemically related to the bioactive peptides thymosin alpha 1 and thymopentin. Three peptides were synthesized, Asp-Leu-Lys-Glu-Arg-Lys-Asp-Val-Tyr (3), Arg-Lys-Asp-Val-Tyr-Glu-Glu-Ala-Glu-Asn (2), and Asp-Leu-Lys-Glu-Arg-Lys-Asp-Val-Tyr-Glu-Glu-Ala-Glu-Asn (1), each of which contains the thymopentin sequence and portions of the bioactive sequence of thymosin alpha 1. Peptides 1-3 were assembled from selected blocked fragments that were synthesized by the polymeric-reagent method, using PHBT (polystyrene-bound 1-hydroxybenzotriazole) as the activating polymer. The ability of peptides 1-3 to enhance the activation (RNA synthesis) and proliferation (DNA synthesis) of human T lymphocytes was determined. In comparison to thymosin alpha 1, thymosin alpha 1 (15-28), and thymopentin, peptides 1-3 did not show significant enhancement of these processes.     

Fmoe SPPS using Perloza™ beaded cellulose

Perloza? beaded cellulose was functionalised by a cyanoethylation reduction procedure to give aminopropyl Perloza. Fmoc-amino acids were anchored to aminopropyl Perloza beaded cellulose via the TFA labile 4-oxymethylphenoxyacetyl (HMPA) linker. Using Fmoc-aminoacyl-4-oxymethylphenoxyacetyl-2,4-dichloro-phenyl esters, all 20 amino acids were anchored at substitution levels ranging from 0.37 to 0.65 mmol/g. Fmoc-amino acids were also anchored using the peptide-amide linker 4-[(R,S)-1-[1-(9H-fluoren-9-yl)-methoxycarbonylamino]-(2′,4′dimethoxy-benzyl]phenoxyacetic acid. The Fmoc-aminoacyl resins were used for SPPS using Fmoc chemistry. SPPS was carried out using either an LKB Biolynx 4175 low-pressure pumped column continuous-flow peptide synthesiser or an ABI 430A automated vortexing batchwise instrument. Comparison of peptides made using each synthesiser showed little difference in quality of the crude peptides. Different Fmoc-amino acid activation methods (DIC/HOB_t/DMF, HBTU, DIC/HOB_t/DCM) were found to be equally useful with Perloza. Peptides were cleaved using TFA plus scavengers; however, the TFA-swollen resin was not readily separated from the TFA peptide solution by simple filtration. Therefore alternative cleavage workup procedures were used with Perloza. Peptides were purified by HPLC and characterised by HPLC and amino acid analysis, and in some cases by FAB-MS. Successful syntheses ranged from 5 to 34 amino acids in length. Some of the peptides were also synthesized using a polystyrene support and standardised (ABI Fastmoc?) SPPS protocols. The crude cleaved peptides from each synthesis were compared by HPLC analysis. The overall aim of our work with Perloza is synthesis of resin-bound peptide ligands for affinity chromatography and antibody generation. HPLC analysis of crude peptides showed that peptides up to 20 amino acids in length were of reasonable purity when synthesised using Perloza, thus encouraging us to continue with investigations of SPPS of resin-bound ligands.     

固相法合成心肌兴奋肽及其类似物

用Boc-和Tos-基团分别保护氨基和侧链胍基,以1%交联度聚苯乙烯二苯甲氨基树脂为载体,用DCC固相法合成肽,HF断裂肽树脂键和去除侧链保护基团,粗产物经高效液相层析纯化,合成了心肌兴奋肽Phe-Met-Arg-Phe-NH₂及其类似物Phe-Pro-Arg-Phe-NH₂,并观察了此二种肽对大鼠血压和心率的影响。     

酶解鱼可溶性肽分子组成结构及营养评价

本文叙述了用胃蛋白酶、胰蛋白酶水解的鱼可溶性肤类水解物,经凝胶高效液相色谱和氨基酸分析仪分析测定其肤类分子组成结构和氨基酸组成成分.测得水解产物中肽类相对分子质量在7400以下,其中相对分子质量6600～7400、由52～58个氨基酸组成的较长肽链占1.74%;相对分子质量2500～5300、由20～41个氨基酸组成的中长肽链占29.75%;相对分子质量在1000以下的由2～10个氨基酸组成的寡肽占50%.水解物的总氮与氨基酸态氮比为25.91,约有96%的氨基酸以肽类形式存在,4%为游离氨基酸.测得可溶性肽的总氨基酸含量为73.98%,必需氨基酸为32.39%,占总氮基酸的43.78%,与FAO/WHO相比,苯丙氨酸为第一限制性氨基酸,氨基酸分值为61.根据分析结果,深入探讨了鱼可溶性肽氨基酸组成的平衡性及其有关寡肽在动物机体的生理功能作用.