蛋白质多肽药物制造与临床前研究专栏.doc

2004年第1期蛋白质多肽药物制造与临床前研究专栏重组人组织型纤溶酶原激活剂缺失变体基因的克隆及工程菌的构建与鉴定陈于红 朱镇华 刘蓓钫 张 菁 傅一工王石泉 张 巍 杨 庆 刘建宁（1）重组人组织型纤溶酶原激活剂缺失变体的工业化制备与性质研究朱镇华 孙自勇 陈于红 张 菁 王石泉杨 庆 刘莉莉 丁楅森 陈新园 刘建宁（7）重组人组织型纤溶酶原激活剂缺失变体的药效学研究赵专友 刘厚孝 张 菁 陈于红 张 巍 朱镇华 王石泉 刘建宁（16）一种具有抗肿瘤活性的新型尿激酶原Kringle结构域变体蛋白的研究曹中炜 丁楅森 陈新园 周颖江王石泉 张 菁 朱镇华 陈于红 刘建宁（28）重组人脑钠素在大肠杆菌中的表达、纯化与鉴定孙自勇 朱镇华 陈均勇 刘莉莉 张 巍 杨 庆 张 菁 刘建宁（34）牛肠激酶轻链在毕赤酵母中的分泌表达、纯化和活性鉴定孙自勇 陈均勇 陈新园 汪 晶 田鹏鹏 吴 盛 刘建宁（41）重组人胸腺肽1在大肠杆菌中的表达纯化和鉴定吴 盛 陈新园 陈均勇 刘莉莉 朱镇华 孙自勇 张 燕 刘建宁（49）重组人甲状旁腺激素(1-34)在大肠杆菌中的表达与纯化陈均勇 陈新园 孙自勇 刘莉莉朱镇华 汪 晶 吴 盛 王石泉 刘建宁（58）重组人尿激酶原氨基末端片段的制备及活性测定汪 晶 陈新园 孙自勇 姚宏伟 陈均勇 刘建宁（66）* * * *边无关数为q的n阶树的谱半径的第三大值 郭曙光（75）一类四阶边值问题的n个正解的存在性 姚庆六（83）关于r进制表示法的一个问题数码和问题的探讨 陈凤娟（89）月球多色测光的最新研究进展胡中为 张开均（94）NFZ-10辐照用电子直线加速器束流性能测试高 澎 杭德生 赵明华 肖 林 丁小平（109）光外差速度调制分子离子激光光谱韩良恺 杨晓华 陈扬駸（116）同步12导联ECG的多重分形分布王 俊 宁新宝 陈 颖（124）12导同步心电图波形检测与识别的研究宁新宝 李德华 丁宋威 赵玉成（129）南京大学“国家杰出青年科学基金“获得者介绍支志明(封二)南京大学“国家杰出青年科学基金“获得者介绍李向东(封三)蛋白质多肽药物制造与临床前研究专栏重组人组织型纤溶酶原激活剂缺失变体基因的克隆及工程菌的构建与鉴定 基金项目：国家重大科技专项“创新药物和中药现代化”（2002AA2Z3451） 收稿日期：2003-09-25 通讯联系人：E-mail:jnliu陈于红，朱镇华，刘蓓钫，张 菁，傅一工，王石泉，张 巍，杨 庆，刘建宁（南京大学分子医学研究所，南京，210093）摘 要：利用RT-PCR技术，从人脐带静脉上皮细胞中克隆人组织型纤溶酶原激活剂基因，将其接入TA克隆载体PCR2.1中，经DNA序列测定后，以该重组质粒DNA为模拟，用PCR方法获得了人组织纤容酶原激活剂缺失变体（K2tPA）基因，将其转入pET29a，构建了重组表达质粒pET29a/K2tPA，并转化至大肠杆菌BL21（DE3）中，构成工程菌，经IPTG诱导表达，在40kDa处有一明显表达条带，表达量为约占菌体总蛋白的20%. 该菌种在贮存与复苏及传代过程中具有良好的质粒稳定性和表达稳定性.关键词： 组织型人纤溶酶原激活剂变体基因，工程菌，质粒稳定性，表达稳定性中图分类号： Q 78Gene Cloning, Construction and Characterization of the Engineered Strain of Recombinant Human K2tPAChen Yu-Hong, Zhu Zhen-Hua, Liu Bei-Fang, Zhang Jing, Fu Yi-Gong,Wang Shi-Quan, Zhang Wei, Yang Qing, Liu Jian-Ning(Instituter of Molecular Medicine, Nanjing University, Nanjing, 210093, China)Abstract: K2tPA is one of the third-generation druns. It is a deletion mutation of the wild-type tPA molecule, where the domains of finger, epidermal growth factor and Kringle-1 domains have been removed. We have engineered recombinant human K2tPA. The human tPA gene was obtained from endothelial cells of human umbilical vein by RT-PCR, form which the cDNA of K2tPA was then cloned PCR. The pET29a/K2tPA plasmid was constructed and expressed in E.coli BL21 (DE3) by induction of IPTG. The expression product with 40 kDa was identified by SDS-PAGE, and the protein was found in the inclusion body and accounted for 20% of the total baxterial proteins. It was shown that the engineered strain was very stable for plasmid maintenance, resuscitation, and expression efficiency during storage regeneration.Key words: recombinant human K2tPA, engineered strain, plasmid stability重组人组织型纤溶酶原激活剂缺失变体的工业化制备与性质研究 基金项目：国家重大科技专项“创新药物和中药现代化”（2002AA2Z3451） 收稿日期：2003-09-25 通讯联系人，E-mail:jnliu朱镇华，孙自勇，陈于红，张 菁， 王石泉杨 庆，刘莉莉，丁楅森，陈新园，刘建宁（南京大学分子医学研究所，南京，210093）摘 要： 将含有人组织型纤溶酶原激活剂缺失变体（K2tPA）重组表达质粒的工程菌经10 L种子罐培养及100 L发酵，IPTG诱导表达，其表达量为占菌体总蛋白的20%，表达产物经体外变复性、TI-Sepharose亲和层析、SP-Sepharose离子交换层析，每100 L发酵液得重组人组织型纤溶酶原激活剂缺失变体（K2tPA）纯品4 g， 纯度达95%以上，比活大于500 000IU/mg，内毒素及热源含量、宿主蛋白残留量、宿主DNA残留量等均达到临床使用标准. 其分子量质谱测定）、氨基酸组成、N、C-端氨基酸序列分析等均与理论值相符. 同时进行了等电点测定及肽图分析等性质研究.关键词： 重组人组织型纤溶酶原激活剂缺失变体（K2tPA），发 酵， 活 化，纯 化中图分类号： Q 78Large Scale Preparation and Characterization ofRecombinant Human K2tPAZhu Zhen-Hua, Sun Zi-Yong, Chen Yu-Hong, Zhang Jing, Wang Shi-QuanYang Oing , Liu Li-Li , Ding Bi-Sen, Chen Xin- Yuan , Liu Jian-Ning(Institute of Molecular Medicine, Nanjing University, Nanjing, 210093, China)Abstract: The engineered E. coli containing an expression vector for recombinant human K2tPA was cultured in a 10 L seeding tank, and then grew in a 100 L ferrnentor by induction of IPTG. The expressed protein accounted for 20% of the total bacteria proteins. After renaturation, the folding. solution was applied to an affinity column of TI-Sepharose, and the fraction containing K2tPA activity was further purified by SP-Sepharose ion-exchange chromatography and depynogened by affi-prep polymyxin affinity chromatography. A pilot purification yielded 4 g of purified K2tPA from 100 L medium. The purity of the resulting protein was higher than 95 %, and its specific activity was over 500 000 IU/mg. The trace content of pyrogen, the residual protein and DNA from the host cell all met the requirements for clinical use. The molecular weight weasured by mass spectrum, the amino acid composition and the N/C-terminal analysis of K2tPA were consistent with the theoretical data. The isoelectrophoretic point and peptide mapping of K2tPA were also determined.Key words: human recombinant K2tPA, fermentation, activation, purification重组人组织型纤溶酶原激活剂缺失变体的药效学研究 基金项目：国家重大科技专项“创新药物和中药现代化”（2002AA2Z3451） 收稿日期：2003-09-25 通讯联系人，E-mail:jnliu赵专友1，刘厚孝1，张 菁2，陈于红2，张 巍2，朱镇华2，王石泉2，刘建宁2(1天津药物研究院药理室，天津，300193；2南京大学分子医学研究所，南京，210093)摘 要： 犬静脉给予重组人组织型纤溶酶原激活剂缺失变体(K2tPA)1×106IU/kg、5×105 IU/kg、25×105IU/kg对冠脉血栓产生显著的溶栓效果，栓塞冠脉血管很快出现再通，高、中、低剂量组残存血栓较溶剂对照组分别减少了727、556、425；心肌梗死范围明显缩小血纤维蛋白原明显降解，凝血酶原时间、凝血酶时间及陶土激活部分凝血酶时间明显延长，但抗凝血酶活性无显著改变伤口出血量增加，出血时间延长以上指标与等剂量(5×105IU/kg)的德国Boehringer Mannheim GmbH产品Retavase作用相似.离体家兔血小板凝集实验结果表明：重组人K2tPA对家兔血小板聚集功能具有明显的抑制作用关键词： 重组人K2tPA，溶 栓，纤维蛋白原，血小板聚集中图分类号： R 961A Pharmalogical Study on Recombinant Human K2tPAZhao Zhuan-You1, Liu Hou-Xiao1, Zhang Jing2, Chen Yu-Hong2,Zhang Wei2, Zhu Zhen-Hua2, Wang Shi-Quan2, Liu Jian-Ning2(1. Department of Pharmacology, Tianjing Institute of Medicine, Tianjing, 300193, China;2. Institute of Molecular Medicine, Nanjing University, Nanjing, 210093, China)Abstract: We have generated recombinant human K2tPA. The engineered strain shown in this study was very stable. The purity of the resulting protein was higher than 95%, and its specific activity was over 500 000 IU/ mg. In the current study; recombinant K2tPA administered i.v. to dogs at doses of 1×106 IU/kg, 5×106 IU/ kg and 2.5×106 IU/kg produced obvious effects of coronary thrombolysis, and the coronary artery demonstrated that reperfusion occurred rapidly. The residual thrombi decreased to 72.7 %, 55.6 % and 42.5 % respectively as compared with the control: The areas of myocardial infarction decreased apparently. Fibrinogen degraded significantly. Prothrombin time,thrombin time and activated partial thromplastin time were prolonged significantly, but there was no significant influence on the anti-thrombin activity. The wound bleeding amount increased and the bleeding time was prolonged. In vitro experiment showed that K2tPA inhibited the aggregation activity of platelet. Key words: recombinant human K2tPA, thrombolysis, fibrinogen, platelets aggregation一种具有抗肿瘤活性的新型尿激酶原Kringle 结构域变体蛋白的研究 基金项目：教育部重大科技研究项目（00-03），杰出青年基金（30025011），江苏省“三药”重大科技招标项目（BG2000001）收稿日期：2003-09-25通讯联系人，E-mail:jnliu曹中炜，丁楅森，陈新园，周颖江，王石泉，张 菁，朱镇华，陈于红，刘建宁(南京大学分子医学研究所，南京，210093)摘 要： 利用PCR技术获得人尿激酶原Kringle结构域基因，将其克隆至具有T7启动子的表达质粒pET29a中，并进而插入plasminogen Kringle 5一段16肽片段构建了其变体，重组质粒转化至大肠杆菌BL21中，经IPTG诱导表达，其产物以包涵体形式存在通过体外复性，得到可溶性的prourokinase Kringle及其变体所得蛋白质经过动物肿瘤模型测活，确定变体蛋白具有抑制肿瘤生长作用关键词： 抑止肿瘤生长，Kringle，变 体中图分类号： Q 78A Novel Kringle Mutant of Prourokinase Suppressing Tumor GrowthCao Zhong-Wei, Ding Bi-Sen, Chen Xin-Yuan, Zhou Ying-Jiang,Wang Shi-Quan, Zhang Jing, Zhu Zhen-Hua, Chen Yu-Hong, Liu Jian-Ning(Institute of Molecular Medicine, Nanjing University, Nanjing, 210093, China)Abstract: Kringles of plasminogen and other proteins, obtained by proteolytic fragments, have been reported to display the anti-tumor activity, which represent potent anti-cancer candidates. However, there remains controversy on whether it is the sequence or the tertiary structure that renders Kringle the anti-tumor activity. In order to address such an issue, we cloned the genes of Kringle of prourokinase and obtained its mutant by inserting a previously demonstrated fragment of 16 amino acids from Kringle 5 of plasminogen that manifested anti-turnor activity. The constructed recombinant vectors pET29a were expressed in E. cull BL21 (DE3), induced by IPTG. Prourokinase Kringle and the mutant were first purified by Ni-NTA affinity chromatography and then subjected lo renaturation. Finally,the folding solutions were applied to CM ion-exchange chromatography for further purification and concentration. As a result, appropriately folded proteins with high purity were obtained, which were confirmed by SDS-PAGE analysis. To compare the in vivo anti-tumor activities of prourokinase Kringle and its mutant, male 6-week C57/BL6 mice used for tumor study. Lewis lung carcinoma cells were subcutaneously injected and the anti-efficacy was evaluated on the basis of tumor volume. Here, prourokinase Kringle almost displayed no anti-tumor activity while its mutant comparatively stifled the growth of subcutaneous tumor, illustrating that equipping proteins with certain anti-tumor fragment will inhibit tumor growth and it is the amino acid sequence rather than the tertiary structure of protein that enables several Kringle structures to prevent tumor from growing.Key words: tumor growth suppression, Kringle, mutant重组人脑钠素在大肠杆菌中的表达、纯化与鉴定 基金项目：国家重大科技专项“创新药物和中药现代化”（2002AA2Z3451） 收稿日期：2003-09-25 通讯联系人，E-mail:jnliu孙自勇，朱镇华，陈均勇，刘莉莉，张 巍，杨 庆，张 菁，刘建宁(南京大学分子医学研究所，南京，210093)摘 要： 人脑钠素(hBNP，human brain natriuretic Peptide)是心室分泌的由32个氨基酸组成的多肽激素临床研究表明，hBNP是治疗充血型心衰竭的一种安全有效的多肽药物化学合成脑钠素全基因，以pET32a为表达载体，构建了硫氧还蛋白脑钠素(thioredocin-hBNP)融合蛋白表达质粒，并转化至大肠杆菌BL21(DE3)，经IPTG诱导，融合蛋白的表达量占全菌蛋白量的25以上融合蛋白经肠激酶裂解、Zn-Sepharose亲和层析和C4反相高效液相层析，从每升培养液中可获取4mg rhBNP，纯度达到95经质谱测定，rhBNP样品的分子量为3 464Da体外活性测定结果表明，rhBNP样品对兔胸主动脉条具有显著的血管舒张效应，其ED50为(224±058)×10-6 mg/ml关键词： 脑钠素，肠激酶，酶 解中图分类号： Q78Expression, Purification and Characterization of hBNP in E. coliSun Zi-Yong, Zhu Zhen-Hua, Chen Jun-Yong, Liu Li-Li, Zhang WeiYang Qing , Zhang Jing, Liu Jian-Ning(Institute of Molecular Medicine, Nanjing University, Nanjing, 210093, China)Abstract: B-type or brain natriuretic peptide (BNP), originally isolated from the porcine brain tissue by Sudoh in 1988, is released as preproBNP and then enzymatically cleaved to the N-terminal-proBNP (NT-proBNP) and BNP. Human BNP (hBNP) comprises 32 amino-acids with a central 17 amino-acid ring created by a disulfide bond between Cysl0 and Cys26. Human brain natriuretic peptide is an endogenous peptide hormone secreted by myocardial cells located on both atria and ventricles, primarily by left ventricular myoeardium in response to pressurevolume overloading of the ventricles. Several modulatory mechanisms were involved in the effect of hBNP on systemic blood pressure, such as vasodilating and natriuretic functions, counteracting the vasoconstricting and fluid-retaining effects of the renin-angiotensin-aldosterone system as wdl as regulatory effects on brain nervous system. The counter-regulatory effects of BNP are beneficial in heart failure. Clinical studies have demonstrated that hBNP is a safe and efficient drug for congestive heart failure (CHF).In the present study, the DNA encoding hBNP was chemically synthesized and amplified with polymerase chain reaction (PCR). The amplified hBNP gene was digested with KpnI/Xhol and inserted into the pET32a vector to construct the thioredoxin-hBNP plasmid. The recombinant expression plasmid was transformed into E. coli strain BL21 (DE3), and the expression of thioredoxin-hBNP fusion protein 'was induced with IPTG at a level of 25 % of total host proteins. The fusion protein was digested by enterokinase (EK) to release recombinant hBNP (rhBNP). After the rhBNP was purified by Zn-sepharose affinity chromatography and (24 reverse-phase high-performance liquid chromatography (RP-HPLC), 4 mg rhBNP with a purity of 95% was obtained from 1 liter of bacterial culture. Mass spectrum indicated that the molecular weight of rhBNP was 3 464 Dalton. In vitro biological activity assay revealed that rhBNP significantly induced the relaxation in the rabbit thoracic aortic strip with an EDS0 of (2.24±0.58)×10 -6 mg/mL. Key words: human brain natriuretic peptide, enterokinase, proteolysis牛肠激酶轻链在毕赤酵母中的分泌表达、纯化和活性鉴定 基金项目：国家重大科技专项“创新药物和中药现代化”（2002AA2Z3451） 收稿日期：2003-09-25 通讯联系人，E-mail:jnliu孙自勇，陈均勇，陈新园，汪 晶，田鹏鹏，吴 盛，刘建宁(南京大学分子医学研究所，南京，210093)摘 要： 用RT-PCR扩增肠激酶轻链(EKLC，Enterokinase Light Chain)的cDNA，并克隆至酵母分泌型表达质粒pPICZA中将重组质粒pPICZAEKLC转化至表达宿主pichia pastoris GS 115，经甲醇诱导，目标蛋白EKLC表达并分泌至酵母培养基中采用Zn-Sepharose亲和层析一步纯化，从每升培养液可获得16 mg EKLC，其纯度达90以上酶反应动力学结果表明，EKLC对小分子底物Gly-Asp-Asp-Asp-Asp-Lys-naphthylamide 的Km为(753±42)mol/L，kcat为(315±38)s-1对硫氧化还原蛋白脑钠素融合蛋白(Trx-hBNP)的酶解作用显示，当酶与底物重量比大于1：100时，经37保温5h后，超过75的融合蛋白被裂解上述结果表明，EKLC能够特异识别并水解Asp-Asp-Asp-Asp-Lys肽键，是一种能将融合蛋白中目标蛋白与载体蛋白裂解释放的有效工具酶关键词： 肠激酶，分泌表达，酶 切，免疫印迹中图分类号： Q78Secretory Expression, Purification and Characterization ofBovine Enterokinase Light ChainSun Zi- Yong, Chen Jun-Yong, Chen Xin- Yuan, Wang JingTian Peng-Peng, Wu Sheng, Liu Jian-Ning(Institute of Molecular Medicine, Nanjing University, Nanjing, 210093, China)Abstract: Enterokinase (EK) is a protease at the intestinal brush border that catalyzes the conversion of trypsinogen into active trypsin. This conversion initiates the activation of other pancreatic proteolytic zymogens such as chymotrypsinogen, procarboxypeptidase and proelastase. Enterokinase deficiency seriously impairs protein absorption and leads to severe malabsorption, diarrhoea, and vomiting. Bovine enterokinase is a disulfide-linked heterodimer, composed of an 115 kD heavy chain and a 35 kD light chain. The heavy chain contains an amino- terminal membrane-spanning segment which is involved in substrate recognition and inhibitor specificity. The light chain containing the serine protease domain carries the full catalytic activity. EK is a site-specific protease that cleaves after lysine at the recognition site Asp-Asp-Asp-Asp-Lys. It has been widely used to release recombinant polypeptides from fusion proteins.We describe here the cloning and expression of the bovine enterokinase light chain (EKLC) in the yeast Pichia pastoris. The cDNA encoding EKLC was amplified by RT-PCR and inserted into yeast secretory expression plasmid pPICZA. The plasmid was then transformed into host cells Pichia pastoris GS 115, and after induced with methonal, EKLC was expressed and secreted into the culture medium. 1.6 mg EKLC with a homogeneity greater than 90% was obtained after only a single-step purification with Zn-Sepha