文昌鱼胰岛素样肽的表达、纯化、鉴定及其多克隆抗体制备

为了深入研究胰岛素和胰岛素样生长因子1（IGF－1）的起源和进化以及结构与功能的关系。表达了胰岛素和IGF－1的祖先分子－－文昌鱼胰岛素样肽（ILP）。重组单链ILP的基因用化学方法合成（从cDNA推测的ILPB结构域的C端和A结构域的N端用Ala-Ala-Lys三肽连接起来,并钭B28Arg突变为Lys）,克隆到表达载体pVT102-U中,ILP在酿酒酵母中得到有效表达。发酵液经4步分离纯化,得到均一的单链ILP,经质谱测定分子量和氨基酸组成分析证明表达产物正确。通过Lys-C蛋白内切酶处理将重组单链ILP转化成双链形式。虽然双链ILP与人胰岛素受体没有结合活力。但圆二色性光谱显示它与胰岛素的结构非常相似,用表达的单链ILP免疫新西兰大白兔,获得了高滴度的多克隆抗体。     

基于人磷酸二酯酶4B2高通量筛选异香兰酸芳酰胺抑制剂

磷酸二酯酶4B2(PDE4B2)选择性抑制剂是新型抗炎药物研究的热点。合成异香兰酸芳酰胺衍生物的猪磷酸二酯PDE4强效抑制剂,评价其对人PDE4B2的抑制活性,以明确可用于高通量筛选PDE4抑制剂的靶酶。以异香兰素为原料,经烷基化、氧化、酰胺化反应制得目标化合物;大肠杆菌原核重组表达人PDE4B2;偶联碱性磷酸酶的孔雀绿定磷法测定酶活性,96孔板高通量测定化合物的抑制常数。结果显示,结构确认的5个异香兰酸芳酰胺衍生物纯度超过88%,其对人PDE4B2的抑制常数明显高于对猪主动脉PDE4的抑制常数。不同来源PDE4对异香兰酸芳酰胺衍生物敏感性不同,拟用人PDE4B2为靶酶进行抑制剂高通量筛选。     

(2R,3S,4S)-2-(N,N-二乙氨基)甲酰氧基-3-苯基-4-苯甲酰基-N-甲基-γ-内酰胺的合成及工艺研究

目的：通过对黄皮酰胺全合成中间体（2R,3S,4S）-2-羟基-3-苯基-4-苯甲酰基-N-甲基-γ-内酰胺（化合物A）2位羟基的酯化,提高脂水分配系数（kP）,考察对谷丙转氨酶活性的影响。方法：以化合物A为原料,通过酰化反应合成（2R,3S,4S）-2-（N,N-二乙氨基）甲酰氧基-3-苯基-4-苯甲酰基-N-甲基-γ-内酰胺（化合物B）,重点考察了摩尔比、反应温度、反应时间等条件对反应的影响。化合物B结构已经元素分析、红外光谱、质谱及核磁共振氢谱确证。结果：化合物A和酰化剂以摩尔比2：3,在160℃下反应1h,目标化合物B。收率78．42％。结论：本合成路线及具体反应方法,具有试剂廉价易得、反应条件温和、后处理简便等优点,是一种较为实用的合成方法。     

(2R,3S,4S)-2-(N,N-二乙氨基)甲酰氧基-3-苯基-4-苯甲酰基-N-甲基-Y-内酰胺的合成及工艺研究

目的:通过对黄皮酰胺全合成中间体(2R,3s,4S)-2-羟基-3-苯基-4-苯甲酰基-N-甲基-Y-内酰胺(化合物A)2位羟基的酯化,提高脂水分配系数(1gP),考察对谷丙转氨酶活性的影响。方法:以化合物A为原料,通过酰化反应合成(2R,3S,4S)-2-(N,N-二乙氨基)甲酰氧基-3-苯基-4-苯甲酰基-N-甲基-Y-内酰胺(化合物B),重点考察了摩尔比、反应温度、反应时间等条件对反应的影响。化合物B结构已经元素分析、红外光谱、质谱及核磁共振氢谱确证。结果:化合物A和酰化剂以摩尔比2:3,在160℃下反应1h,目标化合物B,收率78．42%。结论:本合成路线及具体反应方法,具有试剂廉价易得、反应条件温和、后处理简便等优点,是一种较为实用的合成方法。     

小鼠抗人胰岛素受体单克隆抗体的制备、纯化及鉴定

 M11D杂交瘤细胞株是由人胎盘细胞膜纯化所得胰岛素受体免疫BALB/C小鼠后,取其脾细胞与同系小鼠骨髓瘤细胞株NS-1细胞融合所得。该杂交瘤细胞分泌的抗体经ELISA及放射免疫沉淀法证实为胰岛素受体特异的单克隆抗体。该抗体经Protein A-Sepharose亲和层析分离、纯化,SDS-聚丙烯酰胺梯度凝胶电泳鉴定得分子量分别为53000及23000的两条区带,免疫双扩证明为IgGl。该抗体特异地沉淀125Ⅰ-人胎盘细胞膜胰岛素受体,沉淀经SDS-聚丙烯酰胺凝胶电泳后放射自显影得分子量为135000的特异显影带,与胰岛素受体α亚基分子量相同,说明M11D为抗胰岛素受体α亚基的单克隆抗体。     

牛胰多肽与CL／PC脂质体作用后二级结构的变化

用傅立叶变换红外光谱研究了ＢＰＰ与膜作用后其二级结构的变化,通过对红外光谱中酰胺Ｉ谱带进行解卷积、微分及曲线拟合等处理,结果表明,ＢＰＰ与脂膜作用后,其二级结构中α－螺旋成分增多,无规卷曲成分减少。     

诱导回输胰岛素分泌细胞对大鼠糖尿病的疗效

目的:研究胰岛素分泌细胞的体外诱导方法及其对大鼠糖尿病的疗效.方法:分离培养大鼠骨髓干细胞,用尼克酰胺及肠促胰岛素类似物诱导其分化为胰岛素分泌细胞.将24只Wistar大鼠随机分为对照组、糖尿病组和诱导组.后两组建立糖尿病模型,将该胰岛素分泌细胞回输至诱导组体内,监测大鼠体重、血糖(空腹及OGTT 120分血糖)及空腹...     

249个氨基酸的人胰岛素原融合蛋白的表达

选Tac启动子,构建了以牛凝乳酶原B前161个氨基酸的多肽基因与人胰岛素原基因的融合基因的表达质粒pJG202,转化大肠杆菌JMl05,表达受IPTG及温度诱导。高表达时,按电泳扫描计算,表达的融合蛋白可占细胞总蛋白的20－35％。经CNBr裂解,S－磺酸解,初步分离S－磺酸型的人胰岛素原后再使之重组,可分离得人胰岛素原纯品,其氨基酸组成、生物活性与人胰岛素原标准相同。     

付里叶变换红外光谱法研究胰岛素的二级结构

付里叶变换红外光谱法测定了胰岛素的二级结构,讨论了信/噪比、谱解析、曲线拟合和吸收组分的指认对结构测定的影响。测定的胰岛素二级结构与X—光结构分析结果吻合。     

囊瓣亮花木化学成分的研究（Ⅰ）

从番荔枝科亮花木属植物囊瓣亮花木(Phaeanthus saccopetaloides)中分得8个化合物,经光谱解析和理化常数测定,鉴定了其中3个化合物,分别为穆坪马兜铃酰胺,(-)-北美鹅掌楸脂素-C和胡萝卜甙,它们均为从该属植物中首次分得。     

Isosteric and non-isosteric modification of carboxyl groups of papain

Guanidinated mercuri-papain (Gu-papain) was reacted with N-ethylbenzisoxazolium tetrafluoroborate at pH 4.2, 0 degree C, to yield highly reactive N-ethylsalicylamide esters. On varying the amount of reagent applied 2.5-10 carboxyl groups were modified. Appropriate plotting of the data indicated that all 12 groups exposed in the X-ray structure were modified to an extent of 80% in the final preparation, concomitant with a similar loss of activity towards N alpha-benzoyl-L-arginine ethyl ester. The preparations regained complete activity on saponification of the ester groups and removal of some oligomeric material by gel filtration. Considerable activity was recovered when the ester groups were completely replaced by amide groups by subjecting the esters to ammonolysis in 2 M ammonium acetate/ammonia (pH 9.2). The final preparation, after gel filtration, exhibited Km = 57 +/- 1 mM and kcat = 26 +/- 0.2 s-1 towards BAEE (native papain Km = 18 mM and kcat = 26 s-1). It may be concluded that replacement of a bulky modifying group by an isosteric one may cause considerable recovery of activity, emphasizing the importance of isostericity in suppressing the ionizing ability of ionizable groups; furthermore, that a large shift in overall charge, caused by amidation of all accessible carboxyl groups, does not affect the catalytic steps. The absence of effect of side-chain charges on the ion pair in the active site is briefly discussed.     

Insulin receptor internalization and signalling

The insulin receptor kinase (IRK) is a tyrosine kinase whose activation, subsequent to insulin binding, is essential for insulin-signalling in target tissues. Insulin binding to its cell surface receptor is rapidly followed by internalization of insulin-IRK complexes into the endosomal apparatus (EN) of the cell. Internalization of insulin into target organs, especially liver, is implicated in effecting insulin clearance from the circulation. Internalization mediates IRK downregulation and hence attenuation of insulin sensitivity although most internalized IRKs readily recycle to the plasma membrane at physiological levels of insulin. A role for internalization in insulin signalling is indicated by the accumulation of activated IRKs in ENs. Furthermore, the maximal level of IRK activation has been shown to exceed that attained at the cell surface. Using an in vivo rat liver model in which endosomal IRKs are exclusively activated has revealed that IRKs at this intracellular locus are able by themselves to promote IRS-1 tyrosine phosphorylation and induce hypoglycemia. Furthermore, studies with isolated rat adipocytes reveal the EN to be the principle site of insulin-stimulated IRS-1 tyrosine phosphorylation and associated PI3K activation. Key steps in the termination of the insulin signal are also operative in ENs. Thus, an endosomal acidic insulinase has been identified which limits the extent of IRK activation. Furthermore, IRK dephosphorylation is effected in ENs by an intimately associated phosphotyrosine phosphatase(s) which, in rat liver, appears to regulate IRK activity in both a positive and negative fashion. Thus, insulin-mediated internalization of IRKs into ENs plays a crucial role in effecting and regulating signal transduction in addition to modulating the levels of circulating insulin and the cellular concentration of IRK in target tissues.     

Characterization of Streptozotocin-induced Diabetic Rats and Pharmacodynamics of Insulin Formulations

Morphological and functional changes of rat pancreatic islets caused by administration of streptozotocin (STZ) and the bioavailability of insulin formulations administered to STZ-induced diabetic rats with fasting (12 h) or non-fasting were investigated. Islets isolated from normal rats maintained a good three-dimensional structure and the islet yield was 962.5±86.5 islet equivalent number (IEQ, islets converted to an average diameter of 150 μm). In the diabetic group (>500 mg/ml blood glucose), the islet yield was only 44.4±8.3 IEQ and the islet was severely damaged. The minimum reduction of blood glucose of each formulation, such as insulin solution, microcrystal, and insulin microcrystal capsule, was shown to be 11.3, 11.0, and 16.3 mg/dl, respectively, at 6 h in fasting with diabetic rats. These results indicated that the administration of insulin formulations to the fasting groups increased the severe hypoglycemic effect of insulin action more than in non-fasting diabetic rats. The diabetic rat with fasting has a regulatory disorder in maintaining the blood glucose level. Accordingly, the validity of pharmacological availability as an optimal modeling of insulin formulations is best in non-fasting STZ-induced diabetic rats.     

No Decrease in Free IGF‐I with Increasing Insulin in Obesity‐Related Insulin Resistance

Objective: Different facts suggest that the insulin growth factor (IGF)/ insulin growth factor‐binding protein (IGFBP) system may be regulated by factors other than growth hormone. It has been proposed that, in healthy subjects, free IGF‐I plays a role in glucose metabolism. The role of free IGF‐I in glucose homeostasis in insulin resistance is poorly understood. This study was undertaken to evaluate the effects of acute changes in plasma glucose and insulin levels on free IGF‐I and IGFBP‐1 in obese and non‐obese subjects. Research Methods and Procedures: Nineteen lean and 24 obese subjects were investigated. A frequently sampled intravenous glucose tolerance test was performed. Free IGF‐I and IGFBP‐1 were determined at 0, 19, 22, 50, 100, and 180 minutes. Results: Basal free IGF‐I levels tended to be higher and IGFBP‐1 lower in obese than in lean subjects. IGFBP‐1 levels inversely correlated with basal insulin concentration. To determine the effects of insulin on the availability of free IGF‐I and IGFBP‐1, changes in their plasma concentrations were measured during a frequently sampled intravenous glucose tolerance test. After insulin administration, a significant suppression of free IGF‐I at 22% was observed in lean subjects. In contrast, plasma‐free IGF‐I levels remained essentially unchanged in the obese group. The differences between both groups were statistically significant at 100 minutes (p < 0.01) and 180 minutes (p < 0.05). Serum IGFBP‐1 was suppressed to a similar extent in both groups. Discussion: These data suggest that the concentrations of free IGF‐I and IGFBP‐1 are differentially regulated by obesity. Obesity‐related insulin resistance leads to unsuppressed free IGF‐I levels.     

The Insulin Tolerance Test in Morbidly Obese Patients Undergoing Bariatric Surgery

Objective: To assess the effect of massive weight loss in relation to insulin resistance and its correlation to changes in glycemic homeostasis and lipid profile in severely obese patients. Research Methods and Procedures: A prospective clinical intervention study was carried out with 31 morbidly obese women (body mass index: 54.2 ± 8.8 kg/m²) divided into three groups according to their glucose tolerance test: 14 normal, 8 impaired glucose tolerance, and 9 type 2 diabetes. All subjects underwent an insulin tolerance test with intravenous bolus of 0.1 U insulin/kg body weight before silastic ring vertical gastroplasty Roux‐en‐Y gastric bypass surgery, and again at 2, 4, 6, and 12 months postoperatively. Fasting plasma glucose, hemoglobin A1c, and lipid profile were also evaluated. Results: A reduction of 68 ± 15% in initial excess body weight was evident within 1 year. Along with weight loss, the following statistically significant changes were found: an increase in the insulin‐sensitivity index (Kitt) and a decrease in fasting plasma glucose and hemoglobin A1c, most notably in the type 2 diabetes group. An overall improvement in lipid profile was observed in all three groups. Discussion: Bariatric surgery was an effective therapeutic approach for these obese patients because it reduced both weight and insulin resistance, along with improving metabolic parameters. Significant correlations were found between insulin resistance and metabolic improvements. Weight loss after bariatric surgery induced an improvement in metabolic fitness, related to the reduction in insulin resistance over a range of glucose tolerance statuses from normal to diabetic.     

Reactivity of Glu-22(beta) of hemoglobin S for amidation with glucosamine

X-ray diffraction analysis of deoxyhemoglobin S crystals has implicated that a number of carboxyl groups of the protein are present at or near the intermolecular contact regions. The reactivity of these or other carboxyl groups of hemoglobin S for the amidation with an amino sugar, i.e., glucosamine, and the influence of amidation on the oxygen affinity and polymerization have been investigated. Reaction of oxyhemoglobin S at pH 6.0 and 23 degrees C with 20 mM 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and 100 mM [3H]glucosamine for 1 h resulted in an incorporation of nearly two residues of glucosamine per tetramer. The amidation was very specific for the carboxyl groups of globin; the glucosamine was not incorporated into the heme carboxyls. Derivatization of hemoglobin S by glucosamine increased the O2 affinity of the protein but had no influence on either the Hill coefficient or the Bohr effect. Amidation by glucosamine also increased the solubility of deoxyhemoglobin S by about 55%. Tryptic peptide mapping of the modified hemoglobin S indicated that the peptides beta-T3 and beta-T5 contained the glucosamine incorporated into the protein. Sequence analysis of glucosamine-modified beta-T3 and beta-T5 demonstrated that the gamma-carboxyl groups of Glu-22 and Glu-43, respectively, had been derivatized with glucosamine. The residue Glu-43(beta) shows a high selectivity toward glycine ethyl ester also, whereas Glu-22(beta) is not reactive toward this amine.(ABSTRACT TRUNCATED AT 250 WORDS)     

一种新的B链C端去四肽胰岛素的研制方法

报道了将单体胰岛素前体(MIP)经胰蛋白酶和羧肽酶B两步连续酶切获得B链C端去四肽胰岛素(DTI)的方法。MIP由甲醇酵母表达,最高发酵表达量达到150mg/L。发酵液中MIP通过疏水层析,分子筛初步纯化后直接进行酶切,在胰蛋白酶酶切3h后加入抑制剂paminobenzamidine处理15min,然后直接加入羧肽酶B酶切6h,再通过反相柱纯化即可得到纯品DTI,从分子筛到最后DTI,总纯化得率达到77%。按中国药典小白鼠惊厥法测定得DTI的生物活力为22IU/mg,是胰岛素的80%,在Superdex G-75分子筛上测定DTI的解离聚合曲线,证明其是单体。     

脂肪酸酰化修饰胰岛素的纯化

目的：脂肪酸酰化修饰的胰岛素类似物是常见的获得长效胰岛素的手段,但因其反应位点的偏差易产生多种传统纯化工艺较难以分离的杂质。本文探索以聚苯乙烯类有机聚合物填料为固定相纯化脂肪酸酰化修饰的胰岛素方法。方法：经过填料对比,有机溶剂浓度和pH选择后,再经过一系列正交试验优化,HPLC方法检验纯度和含量,质谱分析定性终产品,从而确定最佳条件。结果：经过对不同厂家不同型号的聚苯乙烯类有机聚合物填料的比较,填料选定采用纳微UniPs30．300为最佳,其目的蛋白吸附量比为6．32mg／ml,洗脱率为95．27％。在一系列的梯度实验中证实采用30-50％异丙醇浓度最佳。之后的pH对比得出。pH在4．0以下才能分离得到纯度在95％以上的产品。在以上一系列的单因素实验基础上,最后经过正交实验优化证实最佳的洗脱方案为采用A相为合25mM硫酸铵的0．1M磷酸盐缓冲液,pH3．0;B相为异丙醇流动相,洗脱梯度为30-50％B的10CV洗脱,流速60cm/h。放大验证可以得到纯度大于97．5％,回收率70％以上的结果。结论：此方法具有分离效果好,成本低,易于放大的优点。     

Protein carboxyl amidation increases the potential extent of protein polyethylene glycol conjugation

Chemical coupling of polyethylene glycol (PEG) to therapeutic proteins reduces their immunogenicity and prolongs their circulating half-life. The limitation of this approach is the number and distribution of sites on proteins available for PEGylation (the N terminus and the -amino group of lysines). To increase the extent of PEGylation, we have developed a method to increase the number of PEGylation sites in a model protein, recombinant methionine alpha,gamma-lyase (recombinant methioninase; rMETase), an enzyme cancer therapeutic cloned from Pseudomonas putida. rMETase was first PEGylated with methoxypolyethylene glycol succinimidyl glutarate-5000 with a molar ratio of PEG:rMETase of 15:1. The carboxyl groups of the initially PEGylated protein were then conjugated with diaminobutane, resulting in carboxyl amidation. This reaction was catalyzed by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, a water-soluble carbodiimide. The steric hindrance provided by the PEG chains already coupled to the protein prevented cross-linking between rMETase molecules during the carboxyl amidation reaction. The carboxyl-amidated PEGylated rMETase was hyper-PEGylated at a molar ratio of PEG to PEG-rMETase of 60:1. Biochemical analysis indicated that 13 PEG chains were coupled to each subunit of rMETase after hyper-PEGylation compared with 6-8 PEG chains attached to the non-carboxyl-amidated PEG-rMETase. Approximately 15-20% of the non-PEGylated rMETase activity was retained in the hyper-PEGylated molecule. Immunogenicity of the hyper-PEG-rMETase was significantly reduced relative to PEG-rMETase and rMETase. Initial results suggest that hyper-PEGylation may become a new strategy for PEGylation of protein biologics.     

双时相门冬胰岛素30联合艾塞那肽对血糖控制不佳2型糖尿病的疗效及安全性分析

目的:探讨双时相门冬胰岛素30联合艾塞那肽在口服降糖药物和基础胰岛素血糖控制不佳的2型糖尿病的疗效及安全性。方法:将在我院接受治疗的72例既往使用的口服降糖药联合基础胰岛素治疗血糖控制不佳的2型糖尿病患者随机、平行、开放平分成治疗组(BIAsp30+艾塞那肽治疗,早餐和晚餐前注射BIAsp30和艾塞那肽注射液)和对照组(睡前1次皮下注射甘精胰岛素),两组均与二甲双胍联合用药。比较两组治疗前后8点血糖谱;比较两组日胰岛素用量、BMI、HbA1c以及低血糖发生次数;比较两组不良事件。结果:治疗8周、16周后,两组8个点血糖与治疗前相比均有明显下降,差异有显著性(P0.05);治疗8周后、16周后,治疗组早餐前和早餐后2小时血糖、午餐前和午餐后2小时血糖值分别与对照组的血糖相比,有统计学差异(P0.05);两组之间的晚餐前和晚餐后2小时血糖、睡觉前血糖(晚上10点)和凌晨3点血糖相互比较无显著性差异(P0.05);治疗16周后,每天胰岛素类似物用量、BMI组间比较无统计学意义(P0.05);两组治疗后HbA1c分别与治疗前相比有统计学意义(P0.05),治疗组治疗后HbA1c与对照组治疗后HbA1c相比,差异有显著性(P0.05);两组低血糖发生次数有明显差异(P0.05);两组不良事件次数相互比较无统计学意义(P0.05)。结论:BIAsp30联合艾塞那肽可显著改善基础胰岛素联合OAD血糖控制不佳的2型糖尿病患者的血糖控制,有效控制血糖,并具有良好的安全性。