医用重组人胰岛素及类似物的生产和研究

医用重组人胰岛素及类似物的生产和研究寿思明（河南医科大学病理教研室,郑州４５００５２）王文霞（河南省中医院内一病区,郑州４５０００２很多年来,医用胰岛素都是从猪和牛的胰腺提取生产的。首先成功地生产人胰岛素的方法就是对猪胰岛素的改造。猪胰岛素与天然人胰...     

胰岛素蛋白质工程: [B16Ala]胰岛素和[B26Ala]胰岛素——两个新型的速效人胰岛素

用大动物(猪)降血糖实验证明[B16Ala]胰岛素和[B26Ala]胰岛素是快速降血糖胰岛素. 它们的前体[B16Ala]PIP和[B26Ala]PIP, 在甲醇酵母体系中的分泌表达量分别为650和130 mg/L. 由于它们是新型的分别保留全部和几乎全部胰岛素体内生物活力的速效胰岛素, 且在甲醇酵母表达体系中得到比较高的表达量, 所以具有很好的临床应用前景.     

胰岛素蛋白质工程：[B9Glu]人胰岛素

用基因定位突变方法将胰岛素Ｂ链第９位的Ｓｅｒ改为Ｇｌｕ。获得速效胰岛素─—［Ｂ９Ｇｌｕ］人胰岛素．它的受体结合能力和体内生物活力分别为猪胰岛素的２１％和４０％。     

胰岛素蛋白质工程;[B9Glu,B10Asp]人胰岛素

用缺口双链ＤＮＡ的定向突变方法分别将胰岛素Ｂ链第９和第１０位的Ｓｅｒ和Ｈｉｓ改变为Ｇｌｕ和Ａｓｐ,获得「Ｂ９Ｇｌｕ,Ｂ１０Ａｓｐ」人胰岛素。其受体结合能力为猪胰岛素的３４．４％,而体内活力与猪胰岛素基本相同     

DNA重组技术生产人胰岛素

人胰岛素是用DNA重组技术生产的第一个药物。这个产品的研究始于管理大规模DNA重组的联邦条例制订或DNA重组技术产品的商业开发之前。本文叙述了为争取大规模进行生产的许可与保证重组DNA产品的鉴定及安全所采取的措施。DNA重组技术的基础研究将继续在生命科学研究中发生巨大的影响,而在它在商业上的应用将取决于经济状况与投入的资本的效益。     

重组人白介素6受体功能区片段的功能鉴定

用生物素标记重组人白介素６受体功能区片段ｒＩＬ６Ｒ－２８及其二联体蛋白ｒＩＬ６Ｒ－５３,竞争ＥＬＩＳＡ表明重组蛋白可以与配基ＩＬ－６特异结合,流式细胞术检测结果表明ＩＬ－６与生物素标记的重组蛋白所形成的复合物能够与７ＴＤ１细胞表面的ｇｐ１３０结合,而７ＴＤ１细胞生长分析则表明,重组蛋白可以增强ＩＬ－６对７ＴＤ１细胞的生长刺激作用。     

重组人BMP-2生物活性的鉴定

目的鉴定在大肠杆菌中表达的重组人骨形态发生蛋白2（recombinant human bone morphogeneticprotein-2,rhBMP-2）的生物学活性。方法构建重组人BMP-2的原核表达质粒,在大肠杆菌中诱导表达目的蛋白,纯化复性后,采用细胞及动物实验对其生物活性进行鉴定。结果细胞实验表明,重组人BMP-2具有诱导MC3T3-E1前成骨细胞向成骨细胞分化的特性。动物实验显示,重组人BMP-2在大鼠肌肉内具有诱导异位成骨功能。结论制备的重组人BMP-2具有较好的生物学活性。     

重组人胰岛素的纯化及其性质的初步研究

构建含有人胰岛素原基因的重组载体,并在大肠杆菌表达系统中进行高效表达。表达产物经变性、复性、凝胶过滤纯化后,再经胰蛋白酶和羧肽酶B酶切作用,产物经离子交换层析纯化得到重组人胰岛素,且具有天然生物学活性。     

人肝再生增强因子CXXC活性结构域的研究

人肝再生增强因子(human augmenter of liver regeneration, hALR)蛋白序列中有一段保守的Cys-Xaa-Xaa-Cys (CXXC)氨基酸序列,针对hALRp的CXXC结构,对hALR分别进行C65A和Q88C突变,表达、纯化突变体蛋白。体外检测hALRp和突变体的黄素腺嘌呤二核苷酸(flavin adenine dinucleotide, FAD)辅助的巯基氧化酶活性,hALR-FAD和hALRQ88C-FAD组与对照组比较有显著差异(P<0.05),hALR-FAD和hALRQ88C-FAD组之间无差异;hALRC65A-FAD组与对照组比较无差异。结果显示,通过C65A突变将CXXC结构破坏后,该突变体的巯基氧化酶活性完全丧失;通过Q88C突变增加一个CXXC序列后,该突变体的巯基氧化酶活性较hALR-FAD未见明显增加;同时,FAD不仅是hALRp发挥巯基氧化酶活性必须的辅助因子,而且有助于hALRp突变体蛋白的复性。     

肝再生增强因子的cDNA克隆、表达及表达产物的生物活性研究

以肝部分切除后再生肝组织为起始材料,利用RT-PCR扩增出大鼠肝再生增强因子（ALR）,亚克隆于pGEM-T载体,核苷酸序列测定证实为大鼠ALR;将ALRcDNA亚克隆于pBV220质粒,构建了原核表达栽体,并获高效表达菌株,特异表达蛋白占细菌总蛋白的15％,原核表达的ALR在体外缺乏促进大鼠原代培养肝细胞及SMMC-7721肝癌细胞DNA合成的活性,但在体内1／3肝部分切除模型中可刺激肝细胞DNA合成;ALR在生物学活性方面与肝脏刺激物（HSS）存在一定差别,ALR和HSS应是两种不同的活性因子．ALR还具有促肝损伤修复的作用,对其深入研究可能为临床治疗严重肝病提供有效的药物.     

用飞行时间质谱研究尿素对重组人肝再生增强因子的修饰作用

为研究尿素在变性、复性过程中对重组人肝再生增强因子 (recombinanthumanaugmenterofliverregeneration ,rhALR)的修饰作用及被修饰后蛋白质的结构和生物活性 ,采用基质辅助激光解吸电离飞行时间质谱仪 (MALDI TOF MS)测定蛋白质分子量及以胰蛋白酶酶解后的蛋白质各肽段的分子量 ,验证蛋白质是否被修饰 ,以急性肝损伤动物模型检测被修饰后蛋白质的生物活性。结果包涵体用尿素变性、复性 ,再经纯化获得的rhALR分子量为 30780 ,比理论分子量 30 0 98增加 6 82 ,且含有赖氨酸残基的酶解肽段分子量增加 4 3。采用盐酸胍变性、复性 ,再经纯化获得的rhALR分子量为 30 0 87,与理论值基本吻合。含赖氨酸残基的酶解肽段分子量亦正常。说明尿素可对rhALR蛋白肽链上的赖氨酸残基修饰。活性实验表明虽然尿素分解释放的氰酸盐在变性复性过程中能与蛋白质肽链上赖氨酸的ε氨基结合 ,造成rhALR蛋白质分子量增加 ,但被修饰的rhALR仍能提高四氯化碳致肝损伤小鼠的存活率。     

肝再生增强因子研究进展

肝再生增强因子是新近克隆的蛋白质因子,能特异地刺激肝源细胞的增殖,并对ＣＣl4所引起的急性肝衰竭有效治作用。本文综述了肝再生增强因子的发现、基因克隆及组织分布等。目前已开始了该因子的基因工程产品研制,它有望成为一种治疗肝病的新药。     

人肝再生增强因子在毕赤酵母中的表达、纯化和生物学活性的研究

肝再生增强因子（ALR）是一类胞源性肝细胞生长因子。为在毕赤酵母中分泌表达人肝再生增强因子（rhALR）,以色谱法分离纯化后进行体外活性研究,构建表达载体pPICZαA- ALR,经电穿孔转入毕赤酵母中,用0.5％甲醇诱导表达;重组酵母培养上清经SDS-PAGE电泳和western blot鉴定后表明, rhALR以分子量为30kD的二聚体为主;定量分析结果表明,重组酵母培养上清中rhALR约占总蛋白的66％,表达量约为40mg/L;经DEAE柱和G75柱纯化后,获得的rhALR纯度大于95％,得率为52％;体外生物学活性实验表明,rhALR能明显促进HepG2、SMMC-7721和NIH-3T3细胞的增殖。     

重组人肝再生增强因子对梗阻性黄疸大鼠肝细胞线粒体DNA的保护作用

目的:初步探讨重组人肝再生增强因子(rhALR)保护及改善梗阻性黄疸大鼠肝细胞线粒体功能及肝功能的机制.方法:144只健康wistar大鼠随机分为SHAM组,BDO-RBF组,BDO-RBF-rhALR组,利用荧光定量PCR方法,对各组大鼠肝细胞总mtDNA、缺失型mtDNA进行相对定量检测.结果:胆道梗阻后,肝细胞线粒体总mtDNA拷贝数出现明显下降(P<0.01).BDO-RBF-rhALR组总mtDNA拷贝数下降程度明显低于BDO-RBF组(P<0.05);对于缺失型mtDNA占总mtDNA的百分比,在SHAM组,未检测出缺失型mtDNA,而在BDO-RBF组及BDO-RBF-rhALR组,均可见缺失型mtDNA,BDO-RBF组的缺失型mtDNA占总mtD-NA的百分比明显高于BDO-RBF-rhALR组(P<0.05);在胆道梗阻解除后,BDO-RBF-rhALR组的总mtDNA的拷贝数及缺失型mtDNA修复速度明显快于BDO-RBF组(P<0.05).结论:rhALR可通过保护及修复梗阻性黄疸大鼠肝细胞受损的mtDNA,达到保护及改善梗阻性黄疸大鼠肝细胞线粒体功能及肝功能的目的.     

ALR and Liver Regeneration

Liver possesses the capacity to restore its tissue mass and attain optimal volume in response to physical, infectious and toxic injury. The extraordinary ability of liver to regenerate is the effect of cross-talk between growth factors, cytokines, matrix components and many other factors. In this review we present recent findings and existing information about mechanisms that regulate liver growth, paying attention to augmenter of liver regeneration.     

Augmenter of liver regeneration: Essential for growth and beyond

Liver regeneration is a well-orchestrated process that is triggered by tissue loss due to trauma or surgical resection and by hepatocellular death induced by toxins or viral infections. Due to the central role of the liver for body homeostasis, intensive research was conducted to identify factors that might contribute to hepatic growth and regeneration. Using a model of partial hepatectomy several factors including cytokines and growth factors that regulate this process were discovered. Among them, a protein was identified to specifically support liver regeneration and therefore was named ALR (Augmenter of Liver Regeneration). ALR protein is encoded by GFER (growth factor erv1-like) gene and can be regulated by various stimuli. ALR is expressed in different tissues in three isoforms which are associated with multiple functions: The long forms of ALR were found in the inner-mitochondrial space (IMS) and the cytosol. Mitochondrial ALR (23 kDa) was shown to cooperate with Mia40 to insure adequate protein folding during import into IMS. On the other hand short form ALR, located mainly in the cytosol, was attributed with anti-apoptotic and anti-oxidative properties as well as its inflammation and metabolism modulating effects. Although a considerable amount of work has been devoted to summarizing the knowledge on ALR, an investigation of ALR expression in different organs (location, subcellular localization) as well as delineation between the isoforms and function of ALR is still missing. This review provides a comprehensive evaluation of ALR structure and expression of different ALR isoforms. Furthermore, we highlight the functional role of endogenously expressed and exogenously applied ALR, as well as an analysis of the clinical importance of ALR, with emphasis on liver disease and in vivo models, as well as the consequences of mutations in the GFER gene.     

Augmenter of liver regeneration (ALR) gene therapy attenuates CCl4-induced liver injury and fibrosis in rats

Liver fibrosis represents a process of healing and scarring in response to chronic liver injury. Augmenter of liver regeneration (ALR) has been shown to protect hepatocytes from various toxins. The aim of this study was to investigate the effects of ALR gene therapy on liver injury and fibrosis induced by CCl₄ in rats and further explore the underlying mechanisms. Human ALR expression plasmid was delivered via the tail vein. ALR gene therapy might protect the liver from CCl₄-induced injury and fibrogenesis by attenuating the mitochondrial dysfunction, suppressing oxidative stress, and inhibiting activation of HSCs. This report demonstrated that ALR gene therapy protected against the ATP loss, increased the activity of ATPase, decreased intrahepatic reactive oxygen species level, and down-regulated transforming growth factor-β1, platelet-derived growth factor-BB, and α-smooth muscle actin expression. Following gene transfer liver function tests were significantly improved. In brief, ALR gene therapy might be an effective therapeutic reagent for liver fibrosis with potential clinical applications.     

Augmenter of liver regeneration causes different kinetics of ERK1/2 and Akt/PKB phosphorylation than EGF and induces hepatocyte proliferation in an EGF receptor independent and liver specific manner

Background/Aim

Augmenter of liver regeneration (ALR) is a potent growth factor which supports liver regeneration in experimental animals. The aim of this study was to compare proliferation as well as the kinetics of ERK1/2 and Akt/PKB phosphorylation by recombinant human ALR (rhALR) and EGF in human hepatocytes and extrahepatic cells.

Methods

Kinetics of ERK1/2 and Akt/PKB phosphorylation were determined in primary human hepatocytes (phh) after stimulation with rhALR and EGF. Induction of proliferation was analyzed in phh and several cell lines of hepatic and extrahepatic origin by the MTT and [³H]-thymidine assay.

Results

The kinetics of ERK phosphorylation showed clear differences, whereby rhALR caused a transient and EGF a permanent increase during the observation period of 60 min. For both, Akt and ERK phosphorylation, EGF caused a faster effect with maximal levels observed already after 2 min, whereas rhALR caused maximal phosphorylation between 10 and 15 min. Using the EGF receptor inhibitor AG1478 we provide evidence of an EGF receptor independent induction of proliferation by rhALR. Furthermore, rhALR induced proliferation only in phh and the human liver derived cell lines HepG2 and Chang. In contrast, EGF enhanced proliferation in all analyzed cell types including cell lines of colon, bronchial, pancreatic and gastric origin (SW480, BC1, L36PL and GC1).

Conclusion

rhALR and EGF induce different kinetics of ERK and Akt phosphorylation in human hepatocytes. The mitogenic effect of rhALR is liver specific and seems to be at least partially independent from EGF receptor mediated signaling.