重组抗人活化血小板单链抗体-尿激酶原导向溶栓剂的基因构建及表达

为了获得特异性高的导向溶栓药物,应用PCR技术,得到抗人活化血小板单抗(SZ-51)的Fab′基因片段。再用酶切方法,将Fab′中CH1基因片段替换成合成的连接分子(linker)基因,构建成单链抗体基因,并插入到人尿激酶原分泌肽基因及低分子量单链尿激酶(scu-PA-32k)之间,最终构建成重组抗人活化血小板单链抗体-尿激酶原融合蛋白基因。此融合蛋白基因在昆虫细胞中得到表达。纯化的表达产物SDS-PAGE鉴定,其分子量约为60kD,与预期值相符。其比活为9000IU/mg蛋白。ELISA法初步证明此重组的融合蛋白具有与活化血小板抗原结合特异性。     

低分子量单链尿激酶与血纤蛋白粘附肽融合基因的构建、表达及产物活性分析

低分子量单链尿激酶(scu-PA-32)可通过激活纤溶酶原转变成纤溶酶溶解血栓,其溶栓具有一定的选择性,出血副作用比双链尿激酶小,但缺少对纤维蛋白的特异亲和力;2种血纤蛋白粘附肽(四肽A4;十二肽A12)都可和纤维蛋白特异性地结合,具有抗纤维蛋白单体聚合的功能,并因此有一定的抗血栓形成作用。本研     

血纤蛋白粘附肽与低分子量单链尿激酶融合基因在大肠杆菌中的表达

人工合成了血纤蛋白粘附肽基因,构建了粘附肽与低分子量单链尿激酶cDNA的融合基因,在大肠杆菌中表达了融合基因。融合基因表达产物的抗原性和天然尿激酶相同,并具有尿激酶的溶纤活性和粘附肽的抗纤维蛋白单体聚合的功能。     

鼠抗人纤维蛋白单链抗体-尿激酶杂合基因的构建及其在中国仓鼠卵巢细胞中的表达

采用 DNA重组技术构建了表达鼠抗人纤维蛋白单链抗体与低分子量尿激酶融合基因的真核表达载体。通过磷酸钙共沉淀法 ,将该表达载体转染到中国仓鼠卵巢细胞二氢叶酸还原酶基因缺陷株 ( CHO- dhfr-)中 ,利用选择培养基筛选出稳定表达的细胞株 ,溶解圈法测定融合蛋白的表达水平为每 1 0 6细胞每天 5 8IU。该融合蛋白保留了与纤维蛋白的结合活性和溶解纤维蛋白的溶纤活性。SDS- PAGE,Western印迹法分析证明融合蛋白的相对分子质量约为 70× 1 0 3     

血纤蛋白粘附肽与低分子量单链尿激酶融合产物活性分析

人工合成了血纤蛋白粘附肽基因,构建了粘附肽与低分子量单链尿激酶ｃＤＮＡ的融合基因,在大肠杆菌中表达了融合基因。融合基因表达产物的抗原性和天然尿激酶相同,并具有尿激酶的溶纤活性和粘附肽的抗纤维蛋白单体聚合的功能。     

两种人源化单链抗体-尿激酶融合基因的构建与表达

Ｓｃｕ ＰＡ 32Ｋ是单链尿激酶 (Ｓｃｕ ＰＡ)分子的Ｎ端肽段被水解后的产物 ,其分子量小但具有与Ｓｃｕ ＰＡ相同的体内外生物活性[1] 。在过去的工作中 ,本实验室利用噬菌体表面呈现技术筛选到 1株对人纤维蛋白特异的鼠源单链抗体[2 ] ,并构建了鼠抗人交联纤维蛋白单链抗体—Ｓｃｕ ＰＡ 32Ｋ融合基因。为解决该融合基因在大肠杆菌中的高表达问题 ,通过在大肠杆菌中表达构建的一系列融合基因的缺失突变体 ,初步认定Ｓｃｕ ＰＡ 32Ｋ基因中两个连排的大肠杆菌稀有密码子ＡＧＧ(精氨酸 )是影响该融合基因表达的主要因素。用ＰＣＲ定位诱变法…     

两种人粒酶B嵌合基因的表达及对肿瘤细胞的杀伤作用

通过重组PCR构建了抗HER2单链抗体基因、绿脓杆菌外毒素 (PE)转位肽序列和活性型粒酶B(GrBa)基因相融合的sFv2 3e PEⅡ GrBa基因 ,以及N端包含PE部分转位肽序列的PEⅡ GrBa基因 .将这 2种粒酶B嵌合蛋白基因瞬时转染或稳定转染HeLa细胞及SKBR 3细胞 .通过间接免疫荧光、细胞计数、MTT、ELISA等方法 ,观察到细胞浆中表达的PEⅡ GrBa蛋白直接杀伤其表达细胞 ;而sFv2 3e PEⅡ GrBa表达后被分泌至细胞外 ,对产生它的细胞没有杀伤性 ,但能够特异识别并杀伤HER2阳性肿瘤细胞 .结果表明 ,抗肿瘤表面抗原的抗体能够介导靶向识别 ,转位结构域可以辅助效应分子活化、转位至细胞液并杀伤细胞 ,为肿瘤的靶向治疗提供了新的策略 .     

不同方式构建的单链抗体-碱性磷酸酶融合蛋白的活性分析

为了研究不同方式构建的单链抗体-碱性磷酸酶融合蛋白原核表达后的生物活性差异,并分析其可溶性表达的影响因素,本研究通过基因工程操作构建含有不同类型的单链抗体-碱性磷酸酶融合基因的表达载体,优化原核表达条件并诱导表达,比较分析宿主菌、连接肽、抗体的单体或二聚体等因素对融合蛋白可溶性表达和生物活性的影响。结果显示不同方式构建的单链抗体-碱性磷酸酶融合蛋白的活性差异显著,在单链抗体和碱性磷酸酶之间添加连接肽能够获得更高活性的融合蛋白,而单链抗体二聚体与碱性磷酸酶的融合蛋白的活性明显降低。因此,单链抗体-碱性磷酸酶融合蛋白的可溶性表达和活性与单链抗体及其构建方式有关,而抗体自身的特性起着关键性作用。     

共表达TrxA和DsbC对富含二硫键的异源蛋白在大肠杆菌中表达的影响

以复制子为p15A的质粒pACU184为基础 ,构建了 3种表达硫氧还蛋白 (TrxA)或 和二硫键异构酶 (DsbC)的表达质粒 .经IPTG诱导 ,克隆的DsbC和TrxA都以可溶的形式高表达 .分别将构建的 3种表达质粒与复制子为colE1并克隆有人源化鼠抗人纤维蛋白单链抗体 低分子量尿激酶融合基因 (C6 UK)的表达质粒共转化大肠杆菌XL1 blue ,在 30℃用IPTG诱导表达 .SDS PAGE显示 ,共表达TrxA或DsbC都能导致C6 UK融合蛋白的部分可溶性表达 ,而且同时共表达TrxA和DsbC 2种分子时 ,C6 UK完全以可溶形式表达 ,但表达量降低 .分别用溶圈法和ELISA检测了各种共表达时可溶表达产物的生物活性 .结果显示 ,只有共表达DsbC时才能检测到明显的C6 UK融合蛋白的双功能     

抗CD20单链抗体与力达霉素强化融合蛋白的制备及其抗肿瘤活性

抗体融合蛋白是新一代抗肿瘤抗体药物.CD20在约95%的B细胞非霍奇金淋巴瘤表面过度表达,是治疗B细胞淋巴瘤的理想靶点.力达霉素(LDM)是强效烯二炔抗肿瘤抗生素,目前已进入Ⅱ期临床阶段.采用DNA重组技术,利用大肠杆菌表达体系,制备抗CD20单链抗体与力达霉素辅基蛋白LDP的基因工程融合蛋白scFv-LDP.经纯化和...     

人源化鼠抗人纤维蛋白单链抗体-尿激酶融合蛋白在大肠杆菌中的功能性表达

尿激酶是目前临床上广泛使用的一种有效的溶栓制剂 ,但由于尿激酶缺乏对血栓的特异亲和性 ,导致临床上尿激酶的使用剂量较大 ,容易出现全身性出血等副作用。因而开发对血栓特异的导向溶栓制剂是目前溶栓药研制的一个重要方向。本实验室在以前的工作中 ,利用噬菌体表面呈现技术 ,筛选到一种对人纤维蛋白特异的鼠单链抗体[1 ] ,在对该鼠抗体可变区进行结构模建的基础上[2 ] ,对其进行了人源化改造和体外亲和力成熟 ,得到亲和力和特异性较鼠抗体更好的人源化单链抗体[3 ] 。为研制导向溶栓制剂 ,本实验室构建了人源化单链抗体 低分子量尿激酶的…     

Thrombin and H64A subtilisin cleavage of fusion proteins for preparation of human recombinant parathyroid hormone

Human parathyroid hormone, hPTH, an 84 amino acid polypeptide, was produced intracellularly inEscherichia coli as a fusion protein, linked to the C-terminus of a 15 kD IgG-binding protein. Approximately 100 mg fusion protein was obtained per liter fermentation medium. To test the efficiency of two alternative enzymatic cleavage methods, two fusion proteins differing only in the linker region were constructed. Cleavage of a Phe-Phe-Pro-Arg linker was obtained with bovine thrombin and cleavage of a Phe-Ala-His-Tyr linker with recombinant H64A subtilisin. Both enzmes yielded the correct N-terminus and cleaved their respective linkers quantitatively, although additional internal cleavage sites in hPTH were detected and characterized. The linker cleavage conditions were optimized and hPTH was purified to homogeneity. Thrombin cleavage resulted in a final yield of 5 mg hPTH/L, while H64A subtilisin cleavage was more specific and gave 8 mg/L. The purified recombinant product was identical to native hPTH and exhibited full biological activity in an adenylate cyclase assay.     

三种连接子多肽对抗癌融合蛋白翻译后剪切效果比较研究

IL-24和Smac为靶向癌症的多基因治疗研究中重要的候选基因.在构建IL-24和Smac双基因表达载体过程中,选择一个稳定高效的连接子非常关键.利用三个不同的连接子多肽序列IETD、EEED、F2A构建三个真核表达质粒pcDNA3.1(+)-IL-24-IETD-Smac、pcDNA3.1(+)-IL-24-EEED-Smac、pcDNA3.1(+)-IL-24-F2A-Smac,并联合5-氟尿嘧啶(5-FU)处理研究各连接子介导的融合蛋白剪切效果.上述实验结果表明,在三种IL-24-linker-Smac双基因表达载体中,F2A连接子剪切效果最佳且与caspase活性成正相关.IETD、EEED连接子介导的融合蛋白都有一定的剪切,但IETD/EEED多肽影响了上游IL-24蛋白的半衰期,加速了剪切后IL-24蛋白的泛素化降解.本研究为构建靶向癌症应用的双基因或多基因载体提供了设计参考.     

Development of tissue plasminogen activator specific "on demand cleavable" (odc) linkers for radioimmunotherapy by screening one-bead-one-compound combinatorial peptide libraries

New strategies are needed to protect normal organs from radiation in cancer radioimmunotherapy (RIT). This can be achieved by rapid clearance of radiometal in the circulation after accumulation of radioimmunoconjugates (RIC) in the tumor. Our strategy is to place highly efficient and specific cleavable linkers between radiometal chelates and the tumor targeting agents. Such linkers must be resistant to cleavage by enzymes present in the plasma and the tumor. After radiotargeting agents have accumulated in the tumor, a cleaving agent can be administered "on demand" to cleave a specific linker, resulting in the release of radiometal from the circulating RIC in a form that will have rapid renal clearance. We have selected TNKase, a thrombolytic agent approved for patient use, as our model on-demand cleaving agent. To identify TNKase-specific linkers, we screened fluorescent-quenched random "one-bead-one-compound" (OBOC) combinatorial peptide libraries. d-Amino acid containing peptides that were specific for TNKase but were resistant to cleavage by plasma and tumor-associated proteases were identified. One of these peptide substrates (rqYKYkf) was used to link the DOTA chelate to ChL6, a monoclonal antibody known to target breast cancer. This antibody conjugate was stable in plasma for 7 days while preserving the immunoreactivity to intact tumor cells. The addition of TNKase at clinical achievable plasma level (10 mug/mL) resulted in the release of 28% of the radiometal from the radioimmunoconjugate within 72 h. This lead linker, after further optimization to increase its response to TNKase, may be useful in the development of more effective radioimmunotherapeutic and radioimaging agents.     

Immobilization and affinity purification of recombinant proteins using histidine peptide fusions

A gene fusion approach to simplify protein immobilization and purification is described. A gene encoding the protein of interest is fused to a gene fragment encoding the affinity peptide Ala-His-Gly-His-Arg-Pro. The expressed fusion proteins can be purified using immobilized metal affinity chromatography. A vector, designed to ensure obligate head-to-tail polymerization of oligonucleotide linkers was constructed by in vitro mutagenesis. A linker encoding the affinity peptide, was synthesized and polymerized to two, four and eight copies. These linkers were fused to the 3' end of a structural gene encoding a two-domain protein A molecule, ZZ, and to the 5' end of a gene encoding beta-galactosidase. Fusion proteins, of both types, with zero or two copies of the linker showed little or no binding to immobilized Zn2+, while a relatively strong interaction could be observed for the fusions based on four or eight copies of the linker. Using a pH gradient, the ZZ fusions were found to be eluted from the resin at different pHs depending on the number of the affinity peptide. These results demonstrate that genetic engineering can be used to facilitate purification and immobilization of proteins to immobilized Zn2+ and that the multiplicity of the affinity peptide is an important factor determining the binding characteristics.     

Precise nucleotide sequence modifications with bidirectionally cleaving class-IIS excision linkers

Bidirectionally cleaving blunt-ended DNA linkers have been constructed to generate defined nucleotide sequence modifications. The oligodeoxynucleotides (termed 'excision linkers'), contain two back-to-back recognition sites for class-IIS restriction endonucleases and provide a new instrument for modifying DNA primary structure. Following insertion of these linkers into host DNA, digestion with the cognate class-IIS enzyme results in a cleavage upstream and downstream from the adjoining enzyme recognition sites. Bidirectional cleavage efficiency can be improved by including spacer nucleotides between the two recognition sites. The number of nucleotides removed from or added to the host DNA depends upon the cleavage shift characteristic of the class-IIS enzyme, the design of the linker (including lateral spacer nucleotides to set the cleavage position), and the method used to make blunt ends from staggered ends following excision of the linker. BspMI linkers constructed in this study have been used to generate defined deletions in the ApR and TcR genes of pBR322. BsmI excision linkers are also described.     

Plasmin- and thrombin-accelerated shedding of syndecan-4 ectodomain generates cleavage sites at Lys(114)-Arg(115) and Lys(129)-Val(130) bonds

Syndecans are transmembranous heparan sulfate proteoglycans abundant in the surface of all adherent mammalian cells and involved in vital cellular functions. In this study, we found syndecan-1, -2, -3, and -4 to be constitutively expressed by human umbilical vein endothelial cells. The exposure of the ectodomains of syndecan-1 and -4 to the cell surface and their constitutive shedding into the extracellular compartment was measured by immunoassays. In the presence of plasmin and thrombin, shedding was accelerated and monitored by detection and identification of (35)S-labeled proteoglycans. To elucidate the cleavage site of the syndecan ectodomains, we used a cell-free in vitro system with enzyme and substrate as the only reactants. For this purpose, we constructed recombinant fusion proteins of the syndecan-1 and -4 ectodomain together with maltose-binding protein and enhanced yellow fluorescent protein as reporter proteins attached to the N and C termini via oligopeptide linkers. After protease treatment of the fusion proteins, the electrophoretically resolved split products were sequenced and cleavage sites of the ectodomain were identified. Plasmin generated cleavage sites at Lys(114) downward arrowArg(115) and Lys(129) downward arrowVal(130) in the ectodomain of syndecan-4. In thrombin proteolysates of the syndecan-4 ectodomain, the cleavage site Lys(114) downward arrowArg(115) was also identified. The cleavage sites for plasmin and thrombin within the syndecan-4 ectodomain were not present in the syndecan-1 ectodomain. Cleavage of the syndecan-1 fusion protein by thrombin occurred only at a control cleavage site (Arg downward arrowGly) introduced into the linker region connecting the ectodomain with the enhanced yellow fluorescent protein. Because both plasmin and thrombin are involved in thrombogenic and thrombolytic processes in the course of the pathogenesis of arteriosclerosis, the detachment of heparan sulfate-bearing ectodomains could be relevant for the development of arteriosclerotic plaques and recruitment of mononuclear blood cells to the plaque.     

An analysis of protein domain linkers: their classification and role in protein folding

Recent advances in protein engineering have come from creating multi-functional chimeric proteins containing modules from various proteins. These modules are typically joined via an oligopeptide linker, the correct design of which is crucial for the desired function of the chimeric protein. Here we analyse the properties of naturally occurring inter-domain linkers with the aim to design linkers for domain fusion. Two main types of linker were identified; helical and non-helical. Helical linkers are thought to act as rigid spacers separating two domains. Non-helical linkers are rich in prolines, which also leads to structural rigidity and isolation of the linker from the attached domains. This means that both linker types are likely to act as a scaffold to prevent unfavourable interactions between folding domains. Based on these results we have constructed a linker database intended for the rational design of linkers for domain fusion, which can be accessed via the Internet at http://mathbio.nimr.mrc.ac.uk.     

Expression of bifunctional enzymes with xylose reductase and xylitol dehydrogenase activity in Saccharomyces cerevisiae alters product formation during xylose fermentation.

To enhance metabolite transfer in the two initial sequential steps of xylose metabolism in yeast, two structural genes of Pichia stipitis, XYL1 and XYL2 encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, were fused in frame. Four chimeric genes were constructed, encoding fusion proteins with different orders of the enzymes and different linker lengths. These genes were expressed in Saccharomyces cerevisiae. The fusion proteins exhibited both XR and XDH activity when XYL1 was fused downstream of XYL2. The specific activity of the XDH part of the complexes increased when longer peptide linkers were used. Bifunctional enzyme complexes, analyzed by gel filtration, were found to be tetramers, hexamers, and octamers. No degradation products were detected by Western blot analysis. S. cerevisiae strains harboring the bifunctional enzymes grew on minimal-medium xylose plates, and oxygen-limited xylose fermentation resulted in xylose consumption and ethanol formation. When a fusion protein, containing a linker of three amino acids, was coexpressed with native XR and XDH monomers in S. cerevisiae, enzyme complexes consisting of chimerical and native subunits were formed. The total activity of these complexes showed XR and XDH activities similar to the activities obtained when the monomers were expressed individually. Strains which coexpressed chimerical subunits together with native XR and XDH monomers consumed less xylose and produced less xylitol. However, the xylitol yield was lower in these strains than in strains expressing only native XR and XDH monomers, 0.55 and 0.62, respectively, and the ethanol yield was higher. The reduced xylitol yield was accompanied by reduced glycerol and acetate formation suggesting enhanced utilization of NADH in the XR reaction.