抗菌肽CM4与人可溶性B淋巴细胞因子hsBAFF在大肠杆菌中的融合表达

为进一步探讨抗菌肽CM4的原核表达及其生物学功能,本实验研究了抗菌肽CM4与人可溶性B淋巴细胞刺激因子hsBAFF的融合表达及抗菌肽CM4的生物学活性。运用PCR把B淋巴细胞因子hsBAFF和家蚕抗菌肽CM4进行基因融合,构建了融合表达载体pET28a (+)/CM4-hsBAFF,并在大肠杆菌中获得高可溶性表达的融合靶蛋白,且存在于超声破碎后的上清,经分子筛Sephadex G-75纯化后的重组融合蛋白用SDS-PAGE和Western blot分析鉴定.SDS-PAGE分析表明：可以通过分子筛一步纯化得到融合蛋白,该重组融合蛋白的分子量约22.0 KDa。Western blot结果显示该重组蛋白能与鼠抗人hsBAFF的抗体发生特异性反应.运用基因工程的方法获得CM4-hsBAFF重组融合蛋白,并具有很好的抑菌生物学活性。     

家蚕胚胎中胚胎发育因子基因BmEDFG4结构的验证及其结合蛋白的筛选

【目的】本研究旨在通过寻找家蚕Bombyx mori胚胎发育因子(embryonic development factor, EDF)基因BmEDF G-四链体(G-quadruplex, G4)结构的结合蛋白,进一步探究G4结构调控家蚕胚胎发育的可能作用和机制。【方法】通过圆二色谱(circular dichroism, CD)和凝胶迁移实验(electrophoretic mobility shift assay, EMSA)验证G4序列在体外是否形成G4结构;通过启动子活性实验验证BmEDF启动子区G4结构对BmEDF的表达调控的影响;通过qRT-PCR检测BmEDF在家蚕胚胎发育各时期的表达量变化。通过EMSA联合质谱分析可能与BmEDF的G4结构结合的蛋白,然后将与G4结构结合的2个候选蛋白BmeIF4H和BmADDH分别进行基因克隆、表达和纯化,再通过EMSA实验分别验证候选蛋白BmeIF4H和BmADDH与BmEDF的G4结构结合与否。【结果】CD和EMSA实验都证明BmEDF的G4序列在体外可以形成G4结构。启动子活性实验表明BmEDF G4结构的存在对BmEDF转录表达具有正调控的作用。qRT-PCR结果表明BmEDF在产卵后120 h时表达量显著升高。经原核表达纯化,获得BmeIF4H和BmADDH重组蛋白。EMSA实验表明重组蛋白BmeIF4H在体外与BmEDF的G4结构结合,BmADDH不与BmEDF G4结构结合。【结论】家蚕胚胎中的BmeIF4H蛋白可能与BmEDF的G4结构结合。本研究为解析家蚕胚胎发育的DNA高级结构调控机理提供了实验证据。     

人白介素-4与大肠杆菌二硫键异构酶DsbC在大肠杆菌中的融合表达及羟胺切割后共复性

目的:通过融合表达、羟胺切割、与二硫键异构酶共复性,获得高表达、高纯度、高生物活性的重组人白细胞介素-4(rhIL-4)。方法:将5端引入了羟胺切割位点的hIL-4基因克隆到大肠杆菌二硫键异构酶DsbC的原核表达载体pET-DsbC中,IPTG诱导表达,对包涵体进行纯化,然后在变性条件下经羟胺切割,利用DsbC的分子伴侣功能与hIL-4进行共复性,最后利用阳离子交换层析纯化获得rhIL-4蛋白。结果:融合蛋白DsbC-hIL-4的表达量占细菌总蛋白的40%以上,以包涵体形式存在;纯化后得到的rhIL-4的相对分子量为15×103,与预期一致,电泳纯度达95%;细胞学实验测定其具有良好的生物学活性。结论:通过融合表达的方法可以提高hIL-4的原核表达量;利用共复性的方式极大地提高了hIL-4的复性率和生物活性。     

肺炎克雷伯菌HdeA蛋白的抗酸理化性质鉴定

肺炎克雷伯菌是一种常见的院内条件致病菌,通常定植在人体的呼吸道和胃肠道。但肺炎克雷伯菌是怎样在胃液的强酸条件下存活并进入肠道尚不十分清楚。本研究发现,酸性条件能够诱导肺炎克雷伯菌KpHdeA基因表达上调。克隆KpHdeA基因并将其转化大肠杆菌,获得可溶性的KpHdeA蛋白并利用镍离子亲和层析纯化得到目的蛋白。光散射分析证明,酸性胁迫下,KpHdeA蛋白能够减少酸诱导的醇脱氢酶聚集。荧光实验证明,酸胁迫可以诱导KpHdeA蛋白的疏水基团暴露。圆二色谱实验证明,酸性条件能够诱导KpHdeA蛋白形成无序结构。此外KpHdeA蛋白的表达能提高大肠杆菌的耐酸能力。故推测,KpHdeA蛋白具有分子伴侣活性,并在肺炎克雷伯菌抵抗酸胁迫中起重要作用。     

重组hepcidin的分离纯化及鉴定

建立了重组hepcidin的分离纯化方法,并鉴定其抗菌活性。经金属螯合初步纯化的重组蛋白在cysteine/cystine氧化还原体系中氧化形成二硫键,用变性条件下的凝胶过滤除去多聚体,稀释复性后用于肠激酶酶切反应,得到重组hepcidin。融合蛋白His-hepcidin经氧化、复性后的总收率为50%,纯度大于95%。酶切后所得重组hepcidin经抑菌圈试验检验,对枯草芽孢杆菌具有抗菌活性。LC-ESI-MS与园二色光谱检测显示重组hepcidin与天然hepcidin相对分子质量相同、二级结构相似。     

一种人胰岛素类似物的制备、鉴定与活性研究

采用PCR方法,将人胰岛素分子B链B10位His突变为Glu,在B25和B26位之间插入Glu,构建了［B10 Glu,B25-Glu-B26］胰岛素原融合蛋白基因.利用通用型质粒pBV220构建表达载体,在大肠杆菌DH5α中表达,表达蛋白为包含体形式,约占菌体总蛋白的20%～30%.经过复性、凝胶过滤等步骤得到胰岛素原融合蛋白.用胰蛋白酶和羧肽酶B酶切,经过DEAE离子交换和RP-HPLC纯化得到胰岛素突变体类似物,并经过质谱测定鉴定.凝胶过滤法测定了蛋白质分子自身的缔合性质,圆二色谱（CD）测定了构象的变化.并分别测定了放免活性、受体结合活性及小白鼠低血糖惊厥实验.结果表明,突变体分子缔合性明显下降.放免活性和受体结合活性分别约为标准胰岛素的63.5%和114.4%, 整体活力略高于天然胰岛素.     

一种新型单链抗体血栓导向显像剂的制备和部分性质研究

 利用对血栓部位活化血小板α 颗粒膜蛋白GMP 140有亲和特异性的单克隆抗体SZ5 1的血栓部位导向作用 ,并利用金属硫蛋白的高金属结合能力 ,在基因工程水平构建分子量适中的单链抗体血栓导向显像剂 .1分子单抗SZ5 1单链抗体 (ScFv SZ5 1)与 1分子小鼠金属硫蛋白 (MT Ⅰ )cDNA的重组基因拼接产物 ,在大肠杆菌菌株BL2 1(DE3)pLysS中进行发酵表达 ,重组蛋白HLMT表达量为 4 0mg L发酵液 .该重组蛋白以包涵体形式存在于菌体中 .通过一种有效的包涵体制备方法可获得目的蛋白含量为 84 %的包涵体沉淀 .在优化的条件进行了包涵体的变复性 ,复性液先后经过Q SepharoseFF阴离子交换层析和SephadexG 5 0凝胶过滤层析进行纯化 .SDS PAGE鉴定了重组蛋白纯度达 95 %以上 .HPLC进一步证明了最终纯化产品的均一性 .质谱测定HLMT分子量为 384 31 3,与理论值基本相符 .等电聚焦电泳测定pI为 3 0 .Western印迹结果表明 ,HLMT中单链抗体部分具有与活化血小板α 颗粒膜蛋白GMP 140结合特异性 .原子吸收分光光度计法 (AAS)证明 ,HLMT中金属硫蛋白保持了原金属结合活性 .重组蛋白HLMT可以进一步进行核素标记实验和动物体内血栓显像实验     

T4多聚核苷酸激酶的原核表达、纯化及初步应用

原核表达、纯化T4多聚核苷酸激酶,并尝试将纯化的T4 PNK用于短探针序列的连接。本研究以合成的pseT基因为模板,PCR扩增出带有NdeⅠ和Bam HⅠ位点的目的片段,构建pseT-pET-15b原核表达载体,并转入E. coli ER2566中诱导表达。Ni-Agarose亲和层析柱纯化重组蛋白后,再进行Western blot鉴定。用纯化后再浓缩的T4 PNK参与探针连接反应,并设置商品T4 PNK和阴性对照。PCR扩增成功获得大于900 bp的目的基因片段,原核表达载体pseT-pET-15b构建成功,经诱导表达的重组蛋白分子量大小约为35 kD,Western blotting确认蛋白表达正确,浓缩后的蛋白浓度达到826μg/m L。电泳结果显示,重组T4 PNK在探针连接中效果较好。本研究成功表达并纯化了可溶性的T4多聚核苷酸激酶,且具有较好的活性,该蛋白可进一步用于后续大批量探针连接反应或其他相关研究,具有一定实际应用价值。     

带有穿膜肽重组PTD-KLF4的制备及活性鉴定

KLF4是Kruppel样转录因子(kruppel-like factors, KLF)家族中的一员,是维持胚胎干细胞(embryonic stem cells, ESCs)全能性的重要转录因子。蛋白质转导结构域(protein transd uction domain, PTD)能够携带大分子进入细胞。为了获得具有穿膜功能的重组KLF4蛋白,利用原核表达载体PKYB表达KLF4与PTD的融合蛋白PTD-KLF4,用Ni柱纯化融合蛋白并作Western blotting鉴定。利用异硫氰酸荧光素(fluorescein isothiocyanate, FITC)标记PTD-KLF4检测其穿越中国仓鼠卵巢(chinese hamster ovary, CHO)细胞细胞膜的能力;用荧光共振能量转移(fluorescence resonance energy transfer, FRET)检测重组融合蛋白PTD-KLF4结合目的DNA的活性。结果表明:重组PTD-KLF4可以成功进入细胞、定位细胞核内,穿膜效率为(22.29±2.1)%。重组融合蛋白PTD-KLF4引起细胞形态变化,并具有与目标DNA序列特异结合的能力。重组PTD-KLF4的制备为外源蛋白诱导多能干细胞(induced pluripotent stem cells, IPSCs)奠定基础。     

重组人胸腺素β4二串体蛋白的原核表达、纯化及生物活性

目的:获得具有生物学活性的重组人胸腺素β4二串体蛋白(Tβ4②)。方法:人工合成人Tβ4全长基因,构建Tβ4②基因,并将该基因克隆入载体pET-22b(+)中,转化宿主细胞大肠杆菌BL21(DE3),IPTG诱导表达后,经疏水作用层析纯化重组Tβ4②蛋白,采用Western印迹鉴定表达产物,并对其进行生物活性检测。结果:表达和纯化了Tβ4②蛋白,重组人Tβ4②在体外可促进人脐静脉内皮细胞的增殖和迁移。结论:重组人Tβ4②具有与化学合成Tβ4相同的功能,并有更高的生物学活性。     

EML4-ALK 融合基因在非小细胞肺癌中的研究进展

肺癌是发病率和死亡率最高的恶性肿瘤,分子靶向治疗以其特异性高、副反应轻的特点正日益受到关注。近年来临床研究发现EML4-ALK融合基因是除EGFR突变及KRAS突变之外的另-个重要的酪氨酸激酶抑制剂的作用靶点,该融合基因在年轻、不吸烟或少吸烟、腺癌、无EGFR和KRAS突变的非小细胞肺癌患者中发生率较高,且该融合基因阳性者对酪氨酸激酶抑制剂耐药,对于ALK抑制剂(如克唑替尼)则有良好的治疗反应,关于该药的临床试验表明:总有效率达57%(46例确定为部分缓解,1例确定为完全缓解),估计6个月无进展生存概率为72%,常见的副反应是1、2级胃肠道反应。该基因及该药的发现为非小细胞肺癌患者带来了希望。     

Fusion induced aggregation of model vesicles studied by dynamic and static light scattering

Membrane fusion is a key step in the virus mediated cell fusion. The vesicular dispersion serves as a model system to study the membrane fusion. We employed dynamic and static light scattering to study the fusion of phosphatidylcholine vesicles in the presence of model fusion peptide fragments from the hemagglutinin HA2 protein. The fusion-induced aggregation under the present experimental setup exhibited strong pH dependence, similar to the parental viral protein. Replacement of the glycine residue at the extreme amino terminus by glutamic acid (G1E) abolished fusion activity. The average molecular mass and diameter of vesicular dispersion obtained from static and dynamic light scattering measurements respectively at neutral and acidic pH showed about three fold increase in acidic solution containing wild type fusion peptide. The light scattering data are consistent with lipid mixing results. The present work demonstrates the utility of light scattering as a facile means to monitor the fusion process.     

Transducing properties of a pre-structured α-helical DPT-peptide containing a short canine adenovirus type 2 E4orf4 PP2A1-binding sequence

Background

Induction of the death pathway resulting from the specific interaction of the PP2A₁ phosphatase with adenoviral E4orf4 protein is a promising approach for cancer therapy. With the aim of deregulating tumor pathways, and mimicking E4orf4 anti-cancer signal, we have previously proposed the DPT technology concept, based on design of specific PP1/PP2A interacting penetrating peptides.

Methods

Using biochemical, structural and cell survival experiments, we have characterized new DPT-peptides containing short PP2A binding sequences.

Results

We identified overlapping sequences, located within the N-terminal domain E4orf4_23-46 of canine adenoviral E4orf4 protein, that interact with the PP2A-Bα subunit of PP2A₁ holoenzyme. We characterized DPT-E4orf4₄ and TAT-E4orf4₄, two bi-partite cell penetrating peptides containing the 12 PP2A₁ binding residues of the canine type 2 E4orf4_27-38 sequence, respectively fused to the DPT-sh1 and TAT shuttle sequences. Surprisingly DPT-E4orf4₄, in contrast to inactive TAT-E4orf4₄, adopted a well defined α-helical structure and co-precipitated PP2A₁ from HeLa cell extracts. DPT-E4orf4₄ also internalized streptavidin-HRP and inhibited survival of HeLa cells more efficiently than TAT, TAT-E4orf4₄ or the previously published anti-tumor TAT-derived peptide shepherdin. DPT-E4orf4₄ also efficiently inhibited the survival of human adherent transformed cells, including wild type and p53 mutated colonic HCT116 cells, without affecting survival of human non-transformed fibroblasts.

Conclusions

We characterized the transducing properties of a new α-helical DPT-E4orf4₄ peptide containing a short PP2A-interacting sequence from canine Adenoviral E4orf4 protein.

General significance

Our results suggest that α-helical structured DPT peptides specifically interacting with PP2A could be a valuable anti-cancer drug design scaffold.     

腺病毒E4orf4蛋白诱导肿瘤特异性细胞死亡及其在癌症治疗中的应用

腺病毒E4orf4蛋白由腺病毒早期第4转录区第4开放读码框编码,为一种多功能调节蛋白质,其活性包括下调早期病毒基因表达和下调影响病毒复制的细胞基因表达,调控病毒基因的选择性转录后剪切以影响病毒感染进程等。当E4orf4脱离病毒环境单独表达时,可诱导不依赖p53和胱天蛋白酶途径的癌细胞特异性细胞死亡,而不影响原代细胞的正常生长。这表明E4orf4对癌细胞有特异性的杀伤作用。本文主要介绍了：（1）E4orf4主要蛋白质伴侣（蛋白磷酸酶2A和Src家族激酶）对E4orf4细胞杀伤作用的贡献;（2）E4orf4诱导的细胞死亡独特模式的基本机制及其特点;（3）近年来利用E4orf4治疗癌症的研究。     

Modulating the Structure of EGFR with UV Light: New Possibilities in Cancer Therapy

The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases. EGFR is activated upon binding to e.g. epidermal growth factor (EGF), leading to cell survival, proliferation and migration. EGFR overactivation is associated with tumor progression. We have previously shown that low dose UVB illumination of cancer cells overexpressing EGFR prior to adding EGF halted the EGFR signaling pathway. We here show that UVB illumination of the extracellular domain of EGFR (sEGFR) induces protein conformational changes, disulphide bridge breakage and formation of tryptophan and tyrosine photoproducts such as dityrosine, N-formylkynurenine and kynurenine. Fluorescence spectroscopy, circular dichroism and thermal studies confirm the occurrence of conformational changes. An immunoassay has confirmed that UVB light induces structural changes in the EGF binding site. A monoclonal antibody which competes with EGF for binding sEGFR was used. We report clear evidence that UVB light induces structural changes in EGFR that impairs the correct binding of an EGFR specific antibody that competes with EGF for binding EGFR, confirming that the 3D structure of the EGFR binding domain suffered conformational changes upon UV illumination. The irradiance used is in the same order of magnitude as the integrated intensity in the solar UVB range. The new photonic technology disables a key receptor and is most likely applicable to the treatment of various types of cancer, alone or in combination with other therapies.     

Four proteins encoded in the gspB-secY2A2 operon of Streptococcus gordonii mediate the intracellular glycosylation of the platelet-binding protein GspB

Platelet binding by Streptococcus gordonii strain M99 is mediated predominantly by the cell surface glycoprotein GspB. This adhesin consists of a putative N-terminal signal peptide, two serine-rich regions (SRR1 and SRR2), a basic region between SRR1 and SRR2, and a C-terminal cell wall anchoring domain. The glycosylation of GspB is mediated at least in part by Gly and Nss, which are encoded in the secY2A2 locus immediately downstream of gspB. This region also encodes two proteins (Gtf and Orf4) that are required for the expression of GspB but whose functions have not been delineated. In this study, we further characterized the roles of Gly, Nss, Gtf, and Orf4 by investigating the expression and glycosylation of a series of glutathione S-transferase-GspB fusion proteins in M99 and in gly, nss, gtf, and orf4 mutants. Compared with fusion proteins expressed in the wild-type background, fusion proteins expressed in the mutant strain backgrounds showed altered electrophoretic mobility. In addition, the fusion proteins formed insoluble aggregates in protoplasts of the gtf and orf4 mutants. Glycan detection and lectin blot analysis revealed that SRR1 and SRR2 were glycosylated but that the basic region was unmodified. When the fusion protein was expressed in Escherichia coli, glycosylation of this protein was observed only in the presence of both gtf and orf4. These results demonstrate that Gly, Nss, Gtf, and Orf4 are all involved in the intracellular glycosylation of SRRs. Moreover, Gtf and Orf4 are essential for glycosylation, which in turn is important for the solubility of GspB.     

棉铃虫核型多角体病毒orf86基因的原核表达、抗体制备与免疫印迹检测

棉铃虫核型多角体病毒(Helicoverpa armigera nucleopolyhedrovirus,HearNPV)orf86是一个功能未知的基因。从HearNPV基因组中通过PCR得到orf86基因编码序列,将其构建于原核表达载体pGEX-4T-2,转化大肠杆菌BL21获得融合表达产物。融合表达蛋白经分离纯化后免疫新西兰大白兔,制备其多克隆抗体。用ELISA测定结果表明,ORF86多克隆抗体效价为1:5.12×105。利用该抗体Western印迹检测HearNPV感染的HZAM1细胞,检测到一个36kD的目的蛋白条带,与ORF86预期大小一致。结果表明该多抗可用于对orf86编码蛋白的检测和相关功能研究。     

Characterization of the cellular localization of C4orf34 as a novel endoplasmic reticulum resident protein

Human genome projects have enabled whole genome mapping and improved our understanding of the genes in humans. However, many unknown genes remain to be functionally characterized. In this study, we characterized human chromosome 4 open reading frame 34 gene (hC4orf34). hC4orf34 was highly conserved from invertebrate to mammalian cells and ubiquitously expressed in the organs of mice, including the heart and brain. Interestingly, hC4orf34 is a novel ER-resident, type I transmembrane protein. Mutant analysis showed that the transmembrane domain (TMD) of hC4orf34 was involved in ER retention. Overall, our results indicate that hC4orf34 is an ER-resident type I transmembrane protein, and might play a role in ER functions including Ca²⁺ homeostasis and ER stress. [BMB Reports 2014; 47(10): 563-568]