蛋白质通量化定位体系的建立及初步应用

目的:对于蛋白质功能而言,蛋白质定位与蛋白质的表达和修饰等同等重要。传统的蛋白质定位一直沿用单个基因、逐个的研究方法,本实验拟建立一种通量蛋白质定位研究体系。方法:采用并优化了细胞微阵列技术,结合绿色荧光蛋白(GFP)标签、激光扫描共聚焦显微镜及反转染技术,用于大规模蛋白质定位研究。结果:初步建立的蛋白质定位微阵列包含107个GFP标记的cDNA表达载体,分别编码107个重要细胞信号传导通路的蛋白质,并与定位数据库中的已知结果进行了比对;对该系统的有效性进行了验证评价。结论:本定位系统可有效地用于通量化蛋白质定位研究,并可以发展用于蛋白质相互作用、泛素-蛋白酶体通路底物筛选等进一步的功能研究。     

Studies of the spontaneous transfer of retinol from the retinol: retinol-binding protein complex to unilamellar liposomes

The transfer of retinol from its complex with the retinol-binding protein to cell surfaces was studied using unilamellar liposomes as a cell surface model. The transfer of retinol to liposomes at 37°C was rapid and reached an apparent equilibrium within 60 min. The amount of retinol transferred to the liposomes at equilibrium was directly proportional to the starting concentration of retinol: retinol-binding protein over a wide range of retinol:retinol-binding protein concentrations and also directly proportional to the concentration of liposomal phospholipid in the system, when the concentration of retinol:retinol-binding protein was held constant. The transfer increased slightly with temperature. Transfer was increased by a factor of 1.8 at pH 4.5 compared to pH around 7. Prealbumin in amounts sufficient to complex all retinol:retinol-binding protein, decreased retinol transfer to liposomes indicating that prealbumin increases the affinity of retinol-binding protein for retinol. Addition of apo retinol-binding protein to the system decreased the transfer of retinol to liposomes considerably probably through competition with the liposomes for retinol. In similarly designed experiments delipidated bovine serum albumin competed much less with liposomes for retinol. The results show that spontaneous transfer of retinol from the retinol:retinol-binding protein complex to liposomal membranes occurs in vitro and suggests that a similar transfer may occur in vivo from retinol:retinol-binding protein to cell surface membranes.     

靶向肽位置影响融合蛋白与细胞结合活性

目的:探讨靶向多肽与标签蛋白(绿色荧光蛋白)的位置关系是否会影响融合蛋白与细胞之间的结合能力。方法:将获得的GE11和LyP1两种靶向多肽分别与增强型绿色荧光蛋白在不同位置融合表达,通过原核系统表达纯化,将纯化的蛋白加入血清饥饿的SMMC-7721肝癌细胞株培养液中,处理3h,通过荧光显微镜观察细胞中绿色荧光蛋白的情况检查融合多肽与细胞的结合情况。结果:绿色荧光蛋白的羧基端和GE11、LyP1多肽分别融合表达,处理细胞后,融合蛋白显示与细胞有很强的结合能力;当GE11、LyP1在绿色荧光蛋白氨基端融合时,融合蛋白几乎不能与细胞结合。在此基础上,检测了多种靶向肽对多种细胞的靶向效应。结论:不合适的融合策略会降低,甚至消除靶向多肽的结合能力;融合大分子量蛋白也会改变靶向肽的靶向效应。因此,当使用靶向多肽携带基因进行研究时,其在融合蛋白中的位置应该非常谨慎。     

Studies of the spontaneous transfer of retinol from the retinol:retinol-binding protein complex to unilamellar liposomes

The transfer of retinol from its complex with the retinol-binding protein to cell surfaces was studied using unilamellar liposomes as a cell surface model. The transfer of retinol to liposomes at 37 degrees C was rapid and reached an apparent equilibrium within 60 min. The amount of retinol transferred to the liposomes at equilibrium was directly proportional to the starting concentration of retinol:retinol-binding protein over a wide range of retinol:retinol-binding protein concentrations and also directly proportional to the concentration of liposomal phospholipid in the system, when the concentration of retinol:retinol-binding protein was held constant. The transfer increased slightly with temperature. Transfer was increased by a factor of 1.8 at pH 4.5 compared to pH around 7. Prealbumin in amounts sufficient to complex all retinol:retinol-binding protein, decreased retinol transfer to liposomes indicating that prealbumin increases the affinity of retinol-binding protein for retinol. Addition of apo retinol-binding protein to the system decreased the transfer of retinol to liposomes considerably probably through competition with the liposomes for retinol. In similarly designed experiments delipidated bovine serum albumin competed much less with liposomes for retinol. The results show that spontaneous transfer of retinol from the retinol:retinol-binding protein complex to liposomal membranes occurs in vitro and suggests that a similar transfer may occur in vivo from retinol:retinol-binding protein to cell surface membranes.     

EpCAM蛋白的表达、纯化及其单克隆抗体的制备

目的:原核表达EpCAM蛋白并制备抗EpCAM特异性单克隆抗体,初步鉴定相应单克隆抗体的特性。方法:PCR扩增EpCAM基因胞外区,将目的基因亚克隆至载体pET-28a(+),转化至大肠埃希菌株BL21,IPTG诱导表达,组氨酸亲和层析法纯化表达产物。纯化蛋白免疫BALB/c小鼠,将成功免疫的小鼠脾细胞与骨髓瘤SP2/0细胞融合,经ELISA筛选得到分泌特异性抗EpCAM的单克隆抗体的细胞株,免疫BALB/c小鼠进一步制备相应的单克隆抗体,并通过Western blot(蛋白质印记)和FACS(流式细胞分析)鉴定单抗的特异性及生物学活性。结果:成功构建重组表达载体pET28a-EpCAM并在大肠杆菌中获得表达,经His-tag亲和层析法获得纯化的EpCAM重组蛋白。EpCAM重组蛋白免疫的BALB/c小鼠的脾细胞与SP2/0细胞融合、筛选,获得两株稳定分泌EpCAM抗体的杂交瘤细胞株,分别命名为4B2、2F2并免疫BALB/c小鼠获得相应的单克隆抗体。Western blot结果显示4B2腹水纯化所得单抗能够识别FaDu细胞系(人咽鳞癌细胞)中的EpCAM蛋白,但2F2未能识别FaDu细胞中的变性的EpCAM蛋白。FACS结果显示两者均能和FaDu细胞中天然的EpCAM蛋白结合。讨论:成功制备了抗EpCAM的单克隆抗体,并能够识别人咽鳞癌细胞系FaDu中表达的EpCAM,为进一步研究EpCAM抗体在肿瘤治疗中的作用提供基础。     

鸡Mx蛋白基因诱变修饰及抗病活性

[目的]进一步研究鸡Mx蛋白第631位氨基酸的变异与鸡群抗病性的相关性.[方法]本实验利用PCR突变技术将鸡Mx蛋白基因的全长cDNA第2032位的碱基由G突变为A(既631位氨基酸的改变),并将突变的Mx基因插入真核表达载体pcDNA3.0,重组表达载体转染COS-Ⅰ细胞后,进行RT-PCR与间接免疫荧光(IFA)鉴定.[结果]对鸡Mx蛋白基因的cDNA进行PCR诱变修饰正确,构建了能够正确表达鸡Mx蛋白的重组真核表达载体;诱变修饰重组Mx蛋白对抗新城疫病毒(NDV)感染分析结果显示,重组Mx蛋白具有较强的抗新城疫病毒生物活性.[结论]为下一步研究鸡Mx蛋白的抗病机理与制备抗病转基因鸡奠定了坚实的基础.     

NOK在骨肉瘤细胞系MG63 EMT过程中的作用

目的:通过对比和检测人正常成骨细胞系hFOB1.19以及骨肉瘤细胞系MG63中NOK以及EMT标志性分子E-cadherin、Vimitin的mRNA和蛋白表达量,并观察NOK对骨肉瘤细胞系MG63中EMT标志性分子E-cadherin及Vimitin的mRNA和蛋白表达量的影响,探讨NOK在骨肉瘤细胞系MG63 EMT过程中的作用。方法:qRT-PCR、Western blot法检测人正常成骨细胞系hFOB1.19以及骨肉瘤细胞系MG63中NOK、E-cadherin、Vimitin的mRNA和蛋白表达量;构建慢病毒并干扰骨肉瘤细胞系MG63中NOK表达,qRT-PCR、Western blot法检测干扰NOK前后EMT标志性分子E-cadherin及Vimitin的mRNA和蛋白表达。结果:相比于人正常成骨细胞系,NOK、Vimitin的mRNA和蛋白在骨肉瘤细胞系MG63中高表达,E-cadherin的mRNA和蛋白在骨肉瘤细胞系MG63中低表达。慢病毒干扰骨肉瘤细胞中NOK表达后,E-cadherin的mRNA和蛋白表达升高,Vimitin的mRNA和蛋白表达降低。结论:NOK具有促进骨肉瘤细胞系MG63发生EMT过程。     

Cloning and partial characterization of Entamoeba histolytica PTPases

Reversible protein tyrosine phosphorylation is an essential signal transduction mechanism that regulates cell growth, differentiation, mobility, metabolism, and survival. Two genes coding for protein tyrosine phophatases, designed EhPTPA and EhPTPB, were cloned from Entamoeba histolytica. EhPTPA and EhPTPB proteins showed amino acid sequence identity of 37%, both EhPTPases showed similarity with Dictyostelium discoideum and vertebrate trasmembranal PTPases. mRNA levels of EhPTPA gene are up-regulated in trophozoites recovered after 96h of liver abscess development in the hamster model. EhPTPA protein expressed as a glutathione S-transferase fusion protein (GST::EhPTPA) showed enzymatic activity with p-nitrophenylphosphate as a substrate and was inhibited by PTPase inhibitors vanadate and molybdate. GST::EhPTPA protein selectively dephosphorylates a 130kDa phosphotyrosine-containing protein in trophozoite cell lysates. EhPTPA gene codifies for a 43kDa native protein. Up-regulation of EhPTPA expression suggests that EhPTPA may play an important role in the adaptive response of trophozoites during amoebic liver abscess development.     

MRP2 蛋白在食管鳞癌中的表达及其与化疗耐药的相关性

目的:探讨MRP2蛋白在食管鳞癌组织中的表达及其与食管癌化疗耐药的关系。方法:收集原发性食管鳞癌手术标本70例,采用免疫组织化学Envision法检测食管鳞癌组织及其癌旁组织中MRP2蛋白的表达情况,并采用MTT法检测食管鳞癌组织对临床常用化疗药物的敏感性,分析其表达与食管癌化疗耐药的关系。结果:70例食管鳞癌组织及其癌旁正常组织中的阳性表达率分别为58.6%及5.0%。MRP2蛋白在食管鳞癌组织中的阳性表达率明显高于癌旁正常食管组织(P<0.01)。食管鳞癌组织对环磷酰胺、5-氟尿嘧啶、吉西他滨、顺铂、卡铂、阿霉素、长春瑞滨、羟喜树碱等化疗药物的敏感性与其相应癌组织中MRP2表达明显相关(P<0.01)。结论:MRP2的表达与食管鳞癌对多种化疗药物耐药有较好的相关性,推测食管鳞癌组织中MRP2的高表达可能对化疗耐药性的发生发展具有促进作用。     

Lipid composition and molecular species of sialic acids of a mouse cell line (JLS-V9) and its ouabain-resistant mutant

Some properties of a mouse cell line (JLS-V9) and its ouabain-resistant mutant clone (JLS-V9OR) were compared. Specific activities of (Na+ + K+)-ATPase (EC 3.6.1.3) of the cell homogenate, particulate fraction and the plasma membrane fraction were 1.12, 1.72 and 8.75 mu mol/h per mg protein, respectively, in the parent cell and 0.97, 1.68 and 9.36 mu mol/h per mg protein in the mutant cell. The half-maximal concentration of ouabain for the inhibition of the (Na+ + K+)-ATPase was 3.4 . 10(-5) M in the parent cell and 4.0 . 10(-4) M in the resistant clone. The contents of phospholipid and cholesterol on a basis of protein in the mutant were 135 and 105% of those in the parent cell. The mutant's monohexosylceramide (HexCer), lactosylceramide (LacCer), trihexosylceramide (GbOse3Cer) and sialyllactosylceramide (GM3) were 111, 145, 274 and 114% of those of the parent cell. Sialic acids of GM3, analyzed by GC-MS, were 98% N-acetylneuraminic and 2% N-glycolylneuraminic acids in the parent cell, whilst 69% N-acetylneuraminic and 31% N-glycolylneuraminic acids in the mutant clone. The in vivo syntheses of these glycolipids were confirmed by the incorporation of radioactive galactose. No significant difference in fatty acid composition was observed between the two cell types. Neutral glycolipids contained mainly 24:0, 24:1, 22:0 and 16:0, whilst in GM3 18:0 and 18:1 predominated. These results show that the lipid content per mg protein is elevated in the ouabain-resistant cell compared to the parent cell.     

Topography of the insertion of LamB protein into the outer membrane of Escherichia coli wild-type and lac-lamB cells

The appearance of newly induced LamB protein at the cell surface of Escherichia coli was followed topographically by immuno-electron microscopy. LamB protein was induced in E. coli wild-type or lac-lamB cells for a short period of time (4 to 6 min), such that the overall level of LamB protein in induced cells was at least twofold higher than that in uninduced cells. Antibodies bound to LamB protein exposed at the cell surface were labeled with a protein A-gold probe, and the probe distribution in briefly induced cells was compared to that in uninduced cells. Analysis of large numbers of cells showed that newly inserted LamB protein appeared homogeneously over the entire cell surface, both in wild-type cells and in lac-lamB cells. A peak of insertion which was observed at the division site of the cell was also observed in the absence of induction and in control experiments in which a nonspecific probe was used. It is concluded therefore that insertion of LamB protein into the cell envelope of E. coli occurs at multiple sites over the entire cell surface. The average amount of LamB protein which appeared at the cell surface after induction was determined for various cell size classes. It was found that cells of various size classes all synthesized LamB protein after induction, indicating that synthesis of the protein was not restricted to cells in a particular stage of the cell cycle. However, the rate of LamB synthesis was found to vary during the cell cycle: this rate was constant regardless of cell size in nondividing cells, whereas it increased in dividing cells. It is concluded that the accumulation of newly induced LamB protein follows a linear pattern.     

Regulation of protein synthesis in lactating rat mammary tissue by cell volume

The effect of changing cell volume on rat mammary protein synthesis has been examined. Cell swelling, induced by a hyposmotic challenge, markedly increased the incorporation of radiolabelled amino acids (leucine and methionine) into trichloroacetic acid (TCA)-precipitable material: reducing the osmolality by 47% increased leucine and methionine incorporation into mammary protein by 147 and 126% respectively. Conversely, cell shrinking, induced by a hyperosmotic shock, almost abolished the incorporation of radiolabelled amino acids into mammary protein: increasing the osmolality by 70% reduced leucine and methionine incorporation into mammary protein by 86 and 93% respectively. The effects of cell swelling and shrinking were fully reversible. Volume-sensitive mammary tissue protein synthesis was dependent upon the extent of the osmotic challenge. Isosmotic swelling of mammary tissue, using a buffer containing urea (160 mM), increased the incorporation of radiolabelled leucine into TCA-precipitable material by 106%. Swelling-induced mammary protein synthesis was dependent upon calcium: removing extracellular calcium together with the addition of EGTA markedly reduced volume-activated protein synthesis. Cell swelling-induced protein synthesis was inhibited by the Ca(2+) ATPase blocker thapsigargin suggesting that volume-sensitive protein synthesis is dependent upon luminal calcium.     

以SELDI芯片进行细胞标本蛋白分析的方法学研究

目的:探讨以SELDI芯片技术进行细胞标本蛋白分析的最适方法及条件,筛选细胞标本蛋白表达差异。方法:对细胞标本分别用超声裂解法,U9细胞裂解缓冲液配方和自配细胞裂解液提取蛋白,以BCA法测定蛋白浓度;分别以磁珠活化后点样和生物芯片处理器点样使蛋白样品与芯片结合;并对提取蛋白进行检测,比较不同蛋白浓度梯度点样及WCX2,SAX2,IMAC-Cu,H50芯片捕获蛋白差异,用WCX2芯片筛选蛋白差异表达。结果:相同培养条件细胞以上述三种不同蛋白提取方法获得的蛋白浓度分别为:0.25±0.034μg/μl,0.6±0.06μg/μl,1.02±0.077μg/μl;生物芯片处理器点样法操作简单,要求样本量较少,点样时间短;SELDI芯片蛋白质峰图谱与蛋白浓度呈较好的正相关;WCX2,SAX2,H50,IMAC-Cu芯片捕获的蛋白质种类有较大区别;在分子量1000～300000Da范围内,以WCX2芯片共检测到87个差异蛋白峰,其中17个呈趋势变化。结论:上述三种方法比较,选用自配的细胞裂解液提取蛋白的浓度较高且更适于芯片研究;生物芯片处理器能较好地使蛋白与芯片结合;SELDI芯片能准确定位蛋白,且其蛋白质峰与被测蛋白浓度呈正相关变化;SELDI各芯片捕获蛋白类型不同,选择适宜芯片或联合运用芯片检测更易获得较理想蛋白差异表达结果。     

Characterization of a protein kinase-phosphoprotein system in free cytoplasmic ribonucleoprotein particles of plasma cell tumours.

A protein kinase, associated with free cytoplasmic ribonucleoprotein particles (free dRNP) has been purified from mouse plasma cell tumours. This protein kinase is able to phosphorylate in vitro endogenous protein from free dRNP. Some characteristics of this protein kinase have been studied. This protein kinase behaves as being cyclic AMP independent. The properties of this protein kinase were compared with other protein kinases: soluble, ribosome-bound, and nuclear protein kinases. Although there are minor differences it is very similar to a ribosome-associated protein kinase from the plasma cell tumours.     

Influence of supramammary lymph node extract on in vitro cell proliferation

Abstract.   Objectives: Experiments were conducted to evaluate whether or not bovine supramammary lymph node extract (LNE) could support cell proliferation when it was substituted for bovine growth serum (BGS) in cell culture media. Materials and Method s: Two different preparations of LNE were tested. The first yielded protein concentration of 3 mg/mL and the second contained 27 mg/mL protein. Three cell lines (MDA-MB-435, MAC-T and 1C6) were used in serum starvation assays to evaluate LNE. Cell proliferation assays were used to determine growth stimulation in the presence of LNE, and short-term or rapid adaptation cultures were evaluated for LNE effects on cell survival. Results : Heat-inactivated preparation 1 supported cell proliferation as well as or better (12–39%) than BGS following 2 days of serum starvation in culture. The second lymph node preparation provided a stimulatory effect (263–702% greater than BGS across all cell lines) following serum starvation at 2.7 and 5.4 mg/mL protein supplementation. A gradual adaptation process with lymph node supplementation into media maintained cell population growth on a short-term basis. However, once cells were trypsinized or scraped and re-seeded into 2.7 mg/mL LNE protein containing media, cells were unable to re-adhere, leaving them detached, and eventually appearing to be dead. Conclusion s: Substitution of BGS with LNE protein dramatically stimulated cells to proliferate, but did not allow for rapid cell population growth adaptation in vitro .     

Delivery of folates to the cytoplasm of MA104 cells is mediated by a surface membrane receptor that recycles

MA104 cells, as well as several other rapidly dividing tissue culture cells, have a folate-binding protein associated with their cell surface. The protein has the properties of a membrane receptor: (a) 5-methyl[3H]tetrahydrofolic acid binds with high affinity (Kd approximately equal to 3 nM); (b) the protein is an integral membrane protein; (c) it appears to deliver physiological concentrations of 5-methyl[3H]tetrahydrofolic acid to the inside of the cell; (d) binding activity is regulated by the concentration of folate within the cell. To better understand the mechanism of action of this receptor, we have studied the pathway of folate internalization. We present evidence that during internalization: (a) folate binds to the membrane receptor; (b) the ligand-receptor complex moves into the cell; (c) the ligand is released from the receptor in an acidic intracellular compartment and moves into the cytoplasm; and (d) the unoccupied receptor returns to the cell surface.     

Protein tyrosine phosphatases as negative regulators of mitogenic signaling

The regulation of tyrosine phosphorylation represents a key mechanism governing cell proliferation. In fibroblasts, inputs from both growth factor and extracellular matrix receptors are required for cell division. Triggering such receptors induces a wave of tyrosine phosphorylation on key signaling molecules, culminating in the activation of cyclin-dependent kinases and cell cycle progression. In general, protein tyrosine kinases stimulate, while protein tyrosine phosphatases inhibit, such cell proliferation pathways. The role of protein tyrosine kinases in mitogenesis has been extensively studied, but the identity and targets of the protein tyrosine phosphatases that regulate cell growth are not well described. In this review, I will survey recent advances in the identification and regulation of protein tyrosine phosphatases that downregulate cell proliferation. J. Cell. Physiol. 180:173–181, 1999. © 1999 Wiley-Liss, Inc.     

Genetic immobilization of cellulase on the cell surface of Saccharomyces cerevisiae

We tried genetically to immobilize cellulase protein on the cell surface of the yeast Saccharomyces cerevisiae in its active form. A cDNA encoding FI-carboxymethylcellulase (CMCase) of the fungus Aspergillus aculeatus, with its secretion signal peptide, was fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast α-agglutinin, a protein involved in mating and covalently anchored to the cell wall. The plasmid constructed containing this fusion gene was introduced into S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The CMCase activity was detected in the cell pellet fraction. The CMCase protein was solubilized from the cell wall fraction by glucanase treatment but not by sodium dodecyl sulphate treatment, indicating the covalent binding of the fusion protein to the cell wall. The appearance of the fused protein on the cell surface was further confirmed by immunofluorescence microscopy and immunoelectron microscopy. These results proved that the CMCase was anchored on the cell wall in its active form. Received: 19 March 1997 / Received revision: 19 May 1997 / Accepted: 1 June 1997