作用力对TNF-α抗原/抗体二维反应的影响

本文研究了作用力对TNF-α抗原/抗体二维反应的影响,结果表明,TNF-α抗原/抗体间的解离力满足正态分布,而非定值,且不同的作用力,回拉速率和接触时间影响抗原/抗体反应的成键概率和键的解离力.

Characterization and expression analysis of TNF-related apoptosis inducing ligand (TRAIL) in grass carp Ctenopharyngodon idella

TRAIL (Apo2 ligand) described as a type II transmembrame protein belonging to the TNF superfamily can induce apoptotic cell death in a variety of cell types. In the present study, a putative cDNA sequence encoding the 299 amino acids of TRAIL (GC-TRAIL) and its genomic organization were identified in grass carp Ctenopharyngodon idella. The predicted GC-TRAIL sequence showed 44 and 41% identities to chicken and human TRAILs, respectively. In a domain search, a tumor necrosis factor homology domain (THD) was identified in the C-terminal portion of TRAILs. The GC-TRAIL gene consists of five exons, with four intervening introns, spaced over approximately 4 kb of genomic sequence. Analysis of GC-TRAlL promoter region revealed the presence of a number of putative transcription factor binding sites, such as Sp1, NF-kappaB, AP-1, GATA, NFAT, HNF, STAT, P53 and IRFI sequences which are important for the expression of other TNF family members. Phylogenetic analysis placed GC-TRAIL and the putative zebrafish (Danio rerio) TRAIL obtained from searching the zebrafish database into one separate cluster near mammalian TRAIL genes, but apart from the reported zebrafish TRAIL-like protein, indicating that the GC-TRAIL is an authentic fish TRAIL. Expression analysis revealed that GC-TRAIL is expressed in many tissues, such as in gills, liver, trunk kidney, head kidney, intestine and spleen. (c) 2005 Elsevier B.V. All rights reserved.

~(12)C~(6+)离子诱导TNFα基因在SMMC-7721细胞中的表达

目的用Polyfect转染试剂将重组质粒pEgr-TNFα转染人肝癌细胞SMMC-7721,研究该重组质粒经12C6+离子作用后在细胞中的表达。方法用ELISA方法检测TNFα的蛋白表达,用RT-PCR方法检测TNFα转录水平的变化。结果转染pEgr-TNFα后在SMMC-7721细胞中TNFα的蛋白表达量为2·06ng/ml,2Gy12C6+离子照射后,表达量增高为2·11ng/ml,明显高于未转染组和转染空载体组;RNA转录水平也明显增高,2Gy12C6+离子照射后增高更明显。结论12C6+离子联合重组质粒pEgr-TNFα,在被转染的SMMC-7721细胞中表达量明显增高。

抗HER2/neu单链抗体增强TNF-α对卵巢癌细胞的抗肿瘤活性

为了探索抗HER2/neu单链抗体与TNF-α联合应用对表面过度表达HER2/neu卵巢癌细胞的生物效应,同时构建了抗HER2/neu单链抗体scFvC6.5的原核表达载体和人TNF-α的原核表达载体,将上述两种重组子分别转化入感受态宿主菌BL21(DE3),得到稳定表达.表达产物主要以包含体形式存在;包含体经过溶解、变性、复性和纯化,得到了分纯的产物.SDS-PAGE和Western-blot检测结果证实,蛋白表达正确.ELISA法验证了scFvC6.5和人TNF-α具有与卵巢癌细胞SKOV-3的结合活性.MTT细胞毒活性试验进一步表明,相对于单独使用TNF-α,联合应用上述两个重组蛋白细胞毒效应明显提高,SKOV-3细胞对TNF-α的敏感性增强.这将为抗HER2/neu抗体的联合抗肿瘤疗法提供一种新的途径,具有潜在的临床应用价值.图5参23

Kinetic analysis of TNF immune complex by using surface plasmon resonane

The kinetic analysis of the interaction between tumor necrosis factor(TNF) and its monoclonal antibody was performed by surface plasmon resonance(SPR) technique. The monoclonal antibody was immobilized to the surface of CM5 sensor chip by amine coupling. TNF at different concentrations was injected across the mAb immobilized surface. The interaction was recorded in real time and could be seen on the sensorgram. One cycle, including association, dissociation and regeneration, lasted no more than 15 min. The interaction results was evaluated using 1 : 1 Langmuir binding model. The kinetic rate constants were calculated to be: k =1.68 X 10(3) L (.) mol(-1) (.) s(-1), k(d) = 1.73 X 10(-4) s(-1), and the affinity constants K-A = 9. 7 X 10(3) L (.) mol(-1), K-r)= 1. 03 X 10(-7) Mol (.) L-1. The X-2 was 3.47, which showed that the interaction is consistent with the 1 : I model. We can see from the results that although there are two binding sites in one mAb molecule, TNF reacts with each site in an independent and noncooperative manner.

Identification and expression of TRAF6 (TNF receptor-associated factor 6) gene in Zhikong Scallop Chlamys farreri

Tumor necrosis factor receptor-associated factor 6 (TRAF6), a key signaling adaptor molecule common to the TNFR superfamily and IL-IR/TLR family, is important not only for a diverse array of physiological processes functions of the TNFR superfamily, but also is involved in adaptive immunity and innate immunity. In this report, the first bivalve TRAF6 (named as CfTRAF6) gene is identified and characterized from Zhikong scallop Chlamys farreri. The full-length cDNA of CfTRAF6 is of 2510 bp, consisting of a 5'-terminal untranslated region (UTR) of 337 bp, a 3'-terminal UTR of 208 bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame (ORF) encoding a polypeptide of 655 amino acids. The predicted amino acid sequence of CfTRAF6 comprises characteristic motifs of the TRAF proteins, including a Zinc finger of RING-type, two Zinc fingers of TRAF-type, a coiled-coil region, and a MATH (the meprin and TRAF homology) domain. The overall amino acid sequence identity between CfTRAF6 and other TRAF6s is 28-68%. Phylogenetic analyses of CfTRAF6 sequence with TRAF sequences from other organisms indicate that CfTRAF6 is a true TRAF6 orthologue. The mRNA expression of CfTRAF6 in various tissues is measured by Real-time RT-PCR. The mRNA transcripts are constitutively expressed in tissues of haemocyte, muscle, mantle, heart, gonad and gill, but the highest expression is observed in the gonad. The temporal expressions of CfTRAF6 mRNA in the mixed primary cultured haemocytes are recorded after treatment with 20 mu g mL(-1) and 0.5 mu g mL(-1) peptido-glycan (PGN). The expression level of CfTRAF mRNA is down-regulated from 1.5 h to 3 h after the treatment with 0.5 mu g mL(-1) PGN, and then recovers to the original level. While the expression of CfTRAF6 is obviously decreased after treatment with 20 mu g mL(-1) PGN, and reach the lowest point (only about 1/9 times to control) at 3 h. The result Suggests that CfTRAF6 can be greatly regulated by PGN and it may be involved in signal transduction and immune response of scallop. (C) 2008 Published by Elsevier Ltd.

Cloning and expression of tumor necrosis factor (TNF alpha) cDNA from red seabream Pagrus major

A fragment of TNFalpha cDNA sequence from red seabream was cloned by homology cloning approach with two degenerated primers which were designed based on the conserved regions of other animals' TNF sequences. The sequence was elongated by 3' and 5' RACE to get the full length CDS sequence. This sequence contained 1264 nucleotides that included a 5' UTR of 85 bp, a 3' UTR of 514 bp and an open reading frame (ORF) of 666 bp which could encode 222 amino acids propeptide. In 3' UTR, there were several mRNA instability motifs and three endotoxin-responsive sequences, but the sequence lacked the polyadenylation signal. The deduced peptide had a clear transmembrane domain, a TNFalpha family signature and a TNF2 family profile. The cell attachment sequence and the glycosaminoglycan attachment sites were also found in the sequence. The red seabream TNF sequence shared relatively high similarity with both mammalian TNFalpha and TNFbeta by multiple sequence alignments. Phylogenetic analysis showed that the piscine TNFalpha were located independently in a different branch compared with mammalian TNFalpha and TNFbeta. Based on the primary and secondary structure analysis and gene expression study, we could concluded that the red seabream TNF should be a TNFalpha, not TNFbeta. RT-PCR was used to study TNFa transcript expression. 24 h after the red seabream was challenged by Vibrio anguillarum, the RS TNFalpha transcript expression were detected in blood, brain, gill, heart, head kidney, kidney, Ever, muscle and spleen. Results showed that TNFalpha mRNA was constitutively expressed in parts of the tissues both in stimulated and unstimulated fish and the expression could be enhanced after the pathogen infection.

Expression of TNF-alpha gene in Anabaena sp PCC 7120 and purification of its product by affinity chromatography

The effects of N (NaNO3) and C (NaAc) source in medium on the expression of tumor necrosis factor-alpha (TNF-alpha) gene in transgenic Anabaena sp. PCC 7120 were compared. The data showed that N source stabilized the expression of foreign protein and C source altered the synthesis of cell walls. Comparing several methods for breaking the cells, supersonic was able to extract TNF-alpha better than others. For purification of TNF-alpha, transgenic Anabaena cells were broken, the extracts were precipitated with ammonia sulfate, and the impure TNF-alpha was eluted from DEAE ion exchange chromatography. Electrophoresis (PAGE-SDS) showed a single band at 17 kD position.

Molecular cloning and characterization of a putative lipopolysaccharide-induced TNF-alpha factor (LITAF) gene homologue from Zhikong scallop Chlamys farreri

LPS-induced TNF-alpha factor (LITAF) is a novel transcriptional factor that was first discovered in LPS-stimulated human macrophage cell line THP-1. LITAF can bind to TNF-a promoter to regulate its expression. The first scallop LITAF (named as CfLITAF) was cloned from Zhikong scallop Chlamys farreri by Expressed Sequence Tag (EST) and Polymerase Chain Reaction (PCR) techniques. The cDNA of CfLITAF was of 1240 bp and consisted of a 5' untranslated region (UTR) of 112 bp, a 3' UTR of 678 bp and an open reading frame (ORF) of 450 bp encoding a polypeptide of 149 amino acids with an estimated molecular mass of 16.08 kDa and theoretical isoelectric point of 6.77. A typical conserved LITAF-domain was identified in CfLITAF by SMART analysis. Homology analysis of the deduced amino acid sequence of CfLITAF with other known sequences by using the BLAST program revealed that CfLITAF was homologous to the LITAF from human and rat (Identity = 46%), cattle, horse, mouse and chicken (Identity = 48%), western clawed frog (Identity = 42%), and zebrafish (Identity = 50%). The mRNA expression of CfLITAF in different tissues including haemocytes, muscle, mantle, heart, gill and gonad, and the temporal expression in haemocytes challenged by LPS or peptidoglycan (PGN) were measured by Real-time RT-PCR. CfLITAF mRNA transcripts could be detected in all tissues examined and be up-regulated in haemocytes after LPS challenge. No significant changes were observed after PGN stimulation. All these data indicated the existence of LITAF in scallop and also provided clue on the presence of TNF-alpha-like molecules in invertebrates. (C) 2007 Elsevier Ltd. All rights reserved.

TNFa在鱼腥藻7120中的高效表达和对宿主光合作用的影响

蓝藻分子生物学的飞速发展已使其成为生物学的前沿。近几年来，以蓝藻为宿主的基因工程发展迅速，使转基因蓝藻已有希望制备药物或处理环境问题。但迄今为止，国内外用蓝藻表达外源基因的表达效率都不高。为了使转基因蓝藻在应用上产生较好的社会效益和经济效益，必须进一步提高外源基因在蓝藻中的表速效率，以及提高光合效率、加速生长。 本研究用人肿瘤坏死因子a（Human Tumor Necrosis Factora简称hTNFa）作为外源目的基因。它是由巨噬细胞和单核细胞受到刺激后产生的一种多功能蛋白质细胞因子。hTNFcc多种生物学效应并作为信号传导体，其中最引人注目的是它对肿瘤组织和肿瘤细胞直接地、特异性和广谱性地杀伤作用，极有希望制成抗癌剩的天然因子之一。但是用大肠杆菌得到的重组产物需要严格纯化，通常用于静脉注射，但由于毒副作用大，十几年来国内外一直停留在临床实验阶段，我们研究组建议用蓝藻为宿主表达hTNFa制备口服剂，来减缓毒副作用，已经得到了转基因鱼腥藻，并测得产物具有抑瘤的生物学活性。但是表达效率一直不高，并且它的表达对蓝藻生长有些抑制。 由于蓝藻是原核生物，基因的表达调控主要是在转录水平和翻译水平。因此，寻找在蓝藻中高效的启动子，改变SD序列的结构是提高外源基因在蓝藻中表达效率的有效手段。本研究将连有不同SD序列的TNFa cDNA克隆到穿梭表达载体pRL-489的启动子（PpsbA）下游，构建2个鱼腥藻7120的穿梭表达载体（pMD-489-TNF1，2），通过三亲接合转移法分别导八鱼腥藻7120细胞。用放射免疫法定量分析TNFa在转基因蓝藻中的表达效率。结果表明，有效地提高了TNFa在鱼腥藻7120中的表达。TNFa的表达量占总可溶性蛋白的2.1 - 2.9%和0.15%，表达效率分别提高到原来的21 - 29倍和1.5倍。 在培养转基因鱼腥藻中，观测到它们在形态和生理上都发生了变化，这反应了TNFcc基因的转入和表达对宿主光合作用的影响。 光学显微镜和扫描电子显微镜的观察发现：转基因鱼腥藻比野生型异形胞数目减少约30%。转入空质粒的营养细胞比野生型略大，转TNFa基因的鱼腥藻异形胞体积明显增加，而营养细胞比正对照和野生型小。到了生长后期，转TNFa因的鱼腥藻营养细胞体积明里增大，多与异形胞相当，有的甚至比异形胞大。转pMD-489-TNFI的鱼腥藻细胞内出现明显的空腔。通过透射电子显微镜的观察发现：转基因藻中的类囊体膜片屡结构更加明显。转基因藻和野生藻的生长曲线的比较表明，转入空质粒pRL-489对宿主的生长几乎没有影响，甚至还略快于野生型;TNFa的表达对细胞的生长有一定副作用，胞内TNFa的含量高时，细胞数增长缓慢，并且平台期时细胞数有一定下降。 从光合作用光强曲线的分析可见，转TNFa因的鱼腥藻有较低的光饱和点，暗示了TNFa的表达可以增强宿主对光的敏感性;同时，TNFa的转入使宿主的呼吸作用加强，几乎比野生型和转空质粒的正对照高一倍，显示了TNFa基因的转入和表达可能给宿主带来更大的代谢负荷;在光饱和点以上，几种藻的真实光合放氧能力大致相同，表明TNFa的表达没有破坏宿主的光合反应中心。 从室温吸收光谱分析可见，转基因蓝藻有相对较高的类胡萝卜素和叶绿素a蓝峰，转TNF谌因的鱼腥藻显示了藻蓝蛋白含量有所降低。因为蓝藻的主要天线色素为藻胆蛋白，藻蓝蛋白相对含量的下降可能与宿主对光更敏感有关。 从低温荧光发射光谱分析可见，转TNFa基因的鱼腥藻7120光系统II能量分配较高。可能是TNFa基因的转入提高了藻胆蛋白的吸收和传递光能的效率。 从叶绿素荧光动力学分析可见，鱼腥藻7120在生长的过程中PSII的活性存在一个变化的过程。TNFa的转入和表达在对数后期提高了宿主的光系统II原初光能转化效率。 从转基因藻光系统I和光系统II光合放氧活性分析与TNFa表达随培养时间变化曲线表明，转TNFcc基因鱼腥藻的光合放氧活性比野生型和正对照高，尤其是显著地提高了宿主的Psn活性。 用自然界中原来不存在的转基因鱼腥藻作上述研究表明：原来只存在于高等、异养的人类和哺乳动物中的TNFa基因，一旦转入最古老的放氧光合生物后，其表达可被调控;同时TNFa的表达又能影响宿主的光合作用。它提高了宿主对光的敏感性、光系统II的活性和对光能的利用率。这似乎都表明TNFa在蓝藻细胞中起信号传导体的作用。而且，这些数据的积累，还有助于我们优化培养条件，提高TNFa的表达效率，为产业化做好准备。

单核细胞、N K细胞和T 细胞在猪-猕猴延迟性异种移植排斥反应中的作用

　目的　观察单核细胞、NK细胞和T 细胞在猪2猕猴延迟性异种移植排斥反应(DXR) 中的作用。方法　建立湖北白猪2云南猕猴的腹腔异位心脏移植模型,实验分为2 组:对照组( n = 5) ,不使用中华眼睛蛇毒因( Y2CVF) ;实验组( n = 4) 应用Y2CVF 完全清除受者体内补体。2 组受 体猴均采用环孢素A(CsA) ,环磷酰胺(CTX) 和甲基强的松龙(MP) 三联免疫抑制治疗。免疫组织 化学方法检测移植心组织中细胞间黏附分子( ICAM)21 、肿瘤坏死因子( TNF)2α、单核细胞、NK 细 胞和T 细胞的表达。结果　对照组3 个移植心在15～60 min 内发生超急性排斥反应(HAR) ,另2 个分别存活22 h 及6 d ,移植心均未见明显的炎性细胞浸润及ICAM21 和TNF2α的表达。实验组 移植心存活时间分别为8 、10 、13 和13 d ,移植物浸润细胞中可见大量的单核细胞(50 %) ,少量的 NK细胞(8 %～10 %) ,CD4 + T 细胞(15 %) 和CD8 + T 细胞(25 %) 。移植物血管内皮细胞表面出现 ICAM21 的表达上调,移植物间质中出现TNF2α的表达增加。结论　单核细胞、NK细胞和T 细胞 介导的移植物损伤,在应用Y2CVF 处理的猪2猕猴DXR 发生中发挥重要作用