重组人核因子κB亚基p65在大肠杆菌中的可溶性表达及纯化

将扩增得到的核因子（NF）κB亚基p65基因片段克隆至测序载体pGEM-T,测序验证该序列为预期目的片段后,再将该基因片段克隆至表达载体pGEX-4T2中,转化大肠杆菌BL21,IPTG诱导表达,用GST蛋白亲和柱纯化融合蛋白p65／GST。Western印迹验证该蛋白具有NF—κB抗原活性。结果表明,构建了NF—κBp65／GST融合表达载体,并表达和纯化了NF—κBp65／GST融合蛋白,为进一步研究NF-κB的功能和筛选NF—κB拮抗分子奠定了基础。     

利用RNA干扰抑制人肝癌细胞中核因子-kB p65基因的表达

目的:用小干扰RNA(siRNA)抑制核因子-KB(NF-kB)p65基因在人肝细胞癌细胞中的表达.方法:从p65的cDNA序列中挑选3个RNA干扰靶位点,用体外转录法制备siRNA,以萤光素酶基因的siRNA为对照,分别转染Hep3B和SMMC-7721细胞,用逆转录半定量PCR和免疫印迹检测siRNA对p65基因表达的抑制效率.结果:3条siRNA对p65基因的表达都有抑制作用,其中2条siRNA的抑制作用更为显著,最高抑制效率约为70%.结论:制备的p65-siRNA可用于研究NF-KB在肝细胞癌发生发展中的作用.     

人类Grx3蛋白与转录因子p65相互作用的研究

巯基氧化还原酶在生物体的系统内担当着非常重要的角色,其主要介导的是巯基和二硫键之间的相互转化的反应。人类Grx3蛋白作为一种巯基氧化还原酶在谷氧还蛋白系统中有着非常重要的作用。已有的研究表明,Grx3蛋白对NF-κB信号通路的活性有十分显著的影响。p65(RELA)是人类机体内十分重要的一种蛋白质,其在NF-κB信号通路有着重要的调控作用,几乎调控着NF-κB信号通路中的所有下游反应。通过大肠杆菌提纯表达的p65与Grx3在体外条件下进行偶联实验,为p65与Grx3存在相互作用提供了强有力的证据,同时也为Grx3蛋白对NF-κB信号通路的影响机制提供了一个可能的解释。     

切胶纯化表达蛋白包涵体的可行性分析

目的:高效率地纯化以包涵体形式表达的蛋白。方法:将SDS-PAGE电泳后的目的蛋白用4mol/L的乙酸钠显现出来,切割下来的目的蛋白可直接用于免疫,将切割下来的目的蛋白装在透析袋中,在电场中将游离的目的蛋白洗脱下来可用于ELISA检测。结果:用该法得到的蛋白经ELISA和Western blot检测均能保持其原有的抗原性,并用该法纯化的蛋白成功制备了相应的多抗和单抗。结论:证实了回收的蛋白仍能保留原有的抗原性,与传统的方法比较,方法简单且比较经济实用特别是包涵体,为切胶纯化蛋白提供了一个新的方法。     

基因工程表达蛋白包涵体的形成和纯化

基因工程表达蛋白包涵体的形成和纯化张庶民,祁自柏综述李河民审（中国药品生物制品检定所,北京１０００５０）在过去的几十年里,基因工程技术的引进给生命科学领域带来了一场革命。科学家们利用基因操作方法克隆出目的基因,然后将其组装入表达质粒,转染到宿主细胞中...     

甲状腺肿瘤p53mRNA及p53蛋白表达的研究

本文采用原位杂交法、免疫组织化学方法分别检测了甲状腺癌ｐ５３ｍＲＮＡ、ｐ５３蛋白的表达,结果显示：２０例甲状腺癌ｐ５３ｍＲＮＡ、ｐ５３蛋白均呈阳性反应,８例甲状腺瘤仅１例呈弱阳性反应,８例Ｇｒａｖｅｓ病全部呈阴性反应。细胞质和细胞核ｍＲＮＡ、ｐ５３蛋白灰度检测发现,甲状腺瘤细胞质、核ｐ５３ｍＲＮＡ灰度值和ｐ５３蛋白灰度值均明显高于Ｇｒａｖｅｓ病,而甲状腺癌其细胞质、核ｐ５３ｍＲＮＡ灰度值和ｐ５３蛋白灰度值又明显高于良性甲状腺瘤,提示甲状腺癌ｐ５３ｍＲＮＡ和ｐ５３蛋白的高表达可能与甲状腺肿瘤细胞分化程度有关     

热休克蛋白HSP65嵌合表达系统的构建和表达

设计一条含(GlySer)3连接肽的引物,从BCG经PCR得到HSP65-Linker的基因片段。将该基因定向克隆入原核表达载体pET42b(+),用大肠杆菌BL21(DE3)转化,构建了pET42-LHSP65的嵌合表达载体。DNA测序正确后经IPTG诱导表达,得到可溶性目的蛋白,Western-blot也证实了此重组蛋白可与抗HSP65发生特异性免疫反应。此重组体的构建为进一步探索HSP65的免疫佐剂功效奠定了基础。     

利用RNA干扰抑制人肝癌细胞中核因子-κB p65基因的表达

目的：用小干扰RNA（siRNA）抑制核因子-κB（NF-κB）p65基因在人肝细胞癌细胞中的表达。方法：从p65的cDNA序列中挑选3个RNA干扰靶位点,用体外转录法制备siRNA,以萤光素酶基因的siRNA为对照,分别转染Hep3B和SMMC-7721细胞,用逆转录半定量PCR和免疫印迹检测siRNA对p65基因表达的抑制效率。结果：3条siRNA对p65基因的表达都有抑制作用,其中2条siRNA的抑制作用更为显著,最高抑制效率约为70%。结论：制备的p65-siRNA可用于研究NF-κB在肝细胞癌发生发展中的作用。     

烟草微丝结合蛋白Fimbrin-ABD1结构域的表达及纯化

微丝骨架在细胞生命活动中发挥着重要作用,微丝结合蛋白通过调控微丝骨架达到调节细胞生理过程的作用,Fimbrin是重要的微丝结合蛋白,目前对其调控机制还不清楚.以烟草Fimbrin基因为模板,首先通过PCR技术获得了其肌动蛋白结合域(actin-binding domain,ABD1)基因,随后构建了蛋白表达载体pET28a-Fimbrin-ABD1,经IPTG诱导获得了Fimbrin-ABD1包涵体,通过摸索包涵体复性的条件得到了有活性Fimbrin-ABD1结构域,并对其功能进行了初步研究,为进一步探讨其作用机制奠定了基础.     

对虾杆状病毒感染螃蟹组织的电镜观察

应用电子显微技术对人工养殖对虾池内的感病螃蟹作了组织切片的电镜观察,发现肝脏、胃、肠组织细胞核内和肌纤维间质中有大量杆状病毒,电镜下的病毒粒子的形态、大小与在中国对虾体内观察到的无病毒包涵体杆状病毒一致。     

大鼠D-双功能蛋白基因原核表达载体的构建及表达

利用原核表达系统构建大鼠D-双功能蛋白表达载体。设计基因拼接引物,通过RT-PCR合成DBP基因cDNA序列,将酶切、纯化的DBP基因与经相同处理的表达载体pET-28 a相连接,转化感受态大肠杆菌DH5α,筛选阳性重组子。将通过酶切及序列分析鉴定阳性的重组子质粒转入感受态大肠杆菌BL21-gold表达菌中,经IPTG诱导表达,通过SDS-PAGE分析目的蛋白表达情况。结果显示,成功获得了包含DBP基因的双链cDNA序列,酶切、序列分析及Western blotting证实成功构建了DBP基因的原核表达载体。通过原核表达系统,DBP蛋白以包涵体形式产生,复性后可获得高表达的目的蛋白。     

Subcellular Distribution of 65,000 Calmodulin-Binding Protein (p65) and Synaptophysin (p38) in Adrenal Medulla

Both neuronal and endocrine cells contain secretory vesicles that store and release neurotransmitters and peptides. Neuronal cells release their secretory material from both small synaptic vesicles and large dense-core vesicles (LDCVs), whereas endocrine cells release secretory products from LDCVs. Neuronal small synaptic vesicles are known to express three integral membrane proteins: 65,000 calmodulin-binding protein (65-CMBP) (p65), synaptophysin (p38), and SV2. A controversial question surrounding these three proteins is whether they are present in LDCV membranes of endocrine and neuronal cells. Sucrose density centrifugation of adrenal medulla was performed to study and compare the subcellular distribution of two of these small synaptic vesicle proteins (65-CMBP and synaptophysin). Subsequent immunoblotting and 125I-Protein A binding experiments performed on the fractions obtained from sucrose gradients showed that 65-CMBP was present in fractions corresponding to granule membranes and intact chromaffin granules. Similar immunoblotting and 125I-Protein A binding experiments with synaptophysin antibodies showed that this protein was also present in intact granules and granule membrane fractions. However, an additional membrane component, equilibrating near the upper portion of the sucrose gradient, also showed strong immunoreactivity with anti-synaptophysin and high 125I-Protein A binding activity. In addition, immunoblotting experiments on purified plasma and granule membranes demonstrated that 65-CMBP was a component of both membranes, whereas synaptophysin was only present in granule membranes. Thus, there appears to be a different subcellular localization between 65-CMBP and synaptophysin in the chromaffin cell.     

高脂喂养大鼠肝脏的NF-κBp65表达与胰岛素抵抗的相关性

目的探讨高脂饲料喂养大鼠肝脏NF-κBp65蛋白的表达与胰岛素抵抗的关系。方法采用高脂饲料喂养建立胰岛素抵抗大鼠模型,并用正常血糖-高血浆胰岛素钳夹技术评估。应用Western blotting方法检测大鼠肝脏中NF-κBp65蛋白的表达。结果①高脂饲料组大鼠的葡萄糖输注率明显低于基础饲料组[GIR60~120（0.76±0.28vs4.26±0.70）mg/（kg.min）,P〈0.01]。②高脂饲料组大鼠肝脏NF-κBp65蛋白的表达明显高于基础饲料组（A值118.48±1.45vs68.13±4.84,P〈0.01）。③高脂胰岛素抵抗大鼠肝脏NF-κBp65蛋白表达与GIR60-120（r=-0.993,P=0.000）和ISI（r=-0.773,P=0.009）负相关。结论高脂诱导的胰岛素抵抗大鼠肝脏NF-κB的激活可能是产生肝脏和全身胰岛素抵抗的根源。     

Preparation and Characterization of Recombinant Murine p65/L-Plastin Expressed in Escherichia coli and High-titer Antibodies against the Protein

We previously identified a 65-kDa protein (p65) that was phosphorylated in activated macrophages. It has turned out to be a murine homologue of human L-plastin, which was identified as a novel protein in human cancer cells. p65/L-plastin is characterized by a series of Ca²⁺-, calmodulin-, and actin-binding domains, and is thought to play a crucial role in leukocytes and cancer cells. We have expressed a recombinant (r) p65/L-plastin in Escherichia coli that binds to β-actin and prepared high-titer antibodies using large amounts of the protein as immunogen. Anti-rp65/L-plastin antibodies recognize native p65/L-plastin as well as rp65/L-plastin and have enabled us to detect the fine structures of intracellular p65/L-plastin, and it was found that its localization was extensively changed by stimulation with bacterial components. We further developed an enzyme-linked immunosorbent assay system and a flow cytometry method using these reagents, which made it possible to measure antibodies, including autoantibodies, against p65/L-plastin and to evaluate the maturation-dependent expression of the protein in leukocytes.     

Cytoplasmic p21 induced by p65 prevents doxorubicin-induced cell death in pancreatic carcinoma cell line

Background

Studies have shown the existence of p21 induction in a p53-dependent and -independent pathway. Our previous study indicates that DOX-induced p65 is able to bind the p21 promoter to activate its transactivation in the cells.

Methods

Over-expression and knock-down experiments were performed in Human Pancreatic Carcinoma (PANC1) cells. Cell cycle and cell death related proteins were assessed by Western Blotting. Cytotoxicity assay was checked by CCK-8 kit. Cell growth was analyzed by flow cytometers.

Results

Here we showed that over-expression of p65 decreased the cytotoxic effect of DOX on PANC1 cells, correlating with increased induction of cytoplasmic p21. We observed that pro-caspase-3 physically associated with cytoplasmic p21, which may be contribution to prevent p21 translocation into the nucleus. Our data also suggested that no clear elevation of nuclear p21 by p65 provides a survival advantage by progression cell cycle after treatment of DOX. Likewise, down-regulation of p65 expression enhanced the cytotoxic effect of DOX, due to a significant decrease of mRNA levels of anti-apoptotic genes, such as the cellular inhibitor of apoptosis-1 (c-IAP1), and the long isoform of B cell leukemia/lymphoma-2 (Bcl-2), leading to efficient induction of caspase-3 cleavage in the cells. More, we present evidence that over-expression of p53 or p53/p65 in the PANC1 cells were more sensitive to DOX treatment, correlated with activation of caspase-3 and clear elevation of nuclear p21 level. Our previous data suggested that expression of p21 increases Gefitinib-induced cell death by blocking the cell cycle at the G1 and G2 phases. The present findings here reinforced this idea by showing p21''s ability of potentiality of DOX-induced cell death correlated with its inhibition of cell cycle progression after over-expression of p53 or p53/p65.

Conclusion

Our data suggested p65 could increase p53-mediated cell death in response to DOX in PANC1 cells. Thus, it is worth noting that in p53 null or defective tumors, targeting in down-regulation of p65 may well be useful, leading to the potentiality of chemotherapeutic drugs.     

HCMV pp65截短蛋白原核表达条件优化

为了提高HCMVpp65蛋白片段在大肠杆菌中表达量,研究了不同发酵条件对其表达的影响,包括培养基、接种量、温度、摇床转速、pH、诱导时间以及诱导剂IPTG使用浓度等。结果表明,以LB培养基为发酵液,按5%接种量,37℃培养3h后IPTG诱导5h,重组菌菌体生物量为0.6g/L,目的蛋白表达量达18mg/L。用3.7L发酵罐进行放大培养,菌体生物量达0.85g/L,最高目的蛋白表达量达到25mg/L。     

重组PLZF蛋白锌指结构域的表达、提纯和活性分析

PLZF(promyelocytic leukaemia zinc finger protein)是一种重要的转录抑制因子,它由位于N端的BTB结构域和C端的锌指结构域构成。鉴于目前对于锌指结构域的立体结构还不是十分清楚,对其进行了高效表达和提纯。为了表达PLZF蛋白的锌指结构域,在其编码序列的5'端加上起始密码ATG后插入到表达载体PET-11a的多克隆位点。构建好的表达质粒转化到BL21 (DE3)大肠杆菌内并用IPTG诱导表达,发现重组蛋白主要以不溶性的包涵体形式在胞内表达。用含有SDS变性剂的缓冲液溶解包涵体后,采用凝胶过滤方法将重组蛋白纯化到纯度达96%以上。对纯化后的蛋白质用反透析的方法进行复性,然后用DNA结合实验进行活性分析,发现复性后的蛋白质具有特异的DNA结合活性,这为进一步研究PLZF蛋白锌指结构域的立体结构打下了重要基础。