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1.
中国HIV-1 B亚型P55和嵌合的P55-V3病毒样颗粒候选疫苗免疫效果的研究 总被引:7,自引:0,他引:7
研究我国艾滋病病毒Ⅰ型(HIV-1)病毒样颗粒(VLP)候选疫苗的免疫原性,为疫苗进行灵长类实验提供实验依据。将gag和gag-V3 VLP不同剂量(50μg、10μg、1μg)在有佐剂(氢氧化铝)和无佐剂条件下皮下免疫小鼠,然后眼眶采血,用ELISA法观察免疫鼠血清中抗体与剂量关系,以及中和抗体滴度和抗体IgG亚型IgG1和IgG2a的水平。同时取鼠脾淋巴细胞,体外抗原刺激后收取淋巴细胞分泌上清 相似文献
2.
纯化的GPI-PLD是分子量为100kD的单肽链,而血清经凝胶过滤时,该酶为500kD。了解酶分子天然状态下的性质,有助于认识其生理功能。采用凝胶过滤、疏水柱层析及超速离心分离并测定该酶活性及磷脂、甘油三酯和胆固醇的浓度。结果说明,GPI-PLD在血清中不是以肽的多聚体形式存在,而可能是与血脂结合,形成蛋白与脂的复合物,经超速离心后存在于HDL的密度区内,但该复合物与富含Apo-A1的HDL亚类是 相似文献
3.
经SephadexG-75凝胶过滤,QAE-SephadexA-50和CM-SephadexC-25离子交换层析的步骤,从湖南产尖吻蝮(Dienagkistrodonacutus)蛇毒中纯化出两个出血毒素(DaHT-1和DaHT-2).SDS-PAGE测得分子量均为23.5kD,IEF-PAGE测得等电点分别为5.6和5.2,两者具有相似的氨基酸组成,其中酸性氨基酸(Asx,Glx)分别占23%和24%,DaHT-1和DaHT-2的最小出血剂量(MHD)分别为0.5μg和0.8μg。都具蛋白水解酶活性,无对TAME,BAEE的水解活性和PLA2酶活性.两者的蛋白水解酶活力与出血活性并非正相关.DaHT-1和DaHT-2的最适温度分别为35℃和40℃,最适pH为6-9,对热均不稳定,温度高于60℃活性完全丧失。金属离子的分析显示每摩尔毒素蛋白约含0.5mol的Zn,1mol的Ca,较多的Na、K、Mg,不含Co。 相似文献
4.
地衣芽孢杆菌1Baciuus Licheniformis)BL-306产生的胞外β-甘露聚糖酶经硫酸铵分级盐析,DEAE-纤维素柱层析。Sephadex-G100柱凝胶过滤和DEAE-纤维素柱再层析分离纯化,得到SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)均一样品。用SDS-PAGE测得纯化后β-甘露聚糖酶分子量为26000道尔顿。用凝胶等电聚焦电泳(PAGEIEF)测得等电点PI为5.0。该酶 相似文献
5.
一种简便快速的蛋白质免疫印渍法介绍 总被引:10,自引:0,他引:10
一种简便快速的蛋白质免疫印渍法介绍骆爱玲,王继伟,李佳格(中国科学院植物研究所,北京100044)ASIMPLEMETHODFORPROTEINBLOTTINGANDLMMUNOASSAYLuoAi-ling;WangJi-wei;Lijia-ge(... 相似文献
6.
从志贺氏1型痢疾村菌LPS中分离纯化出料捩的,以ADH为连接剂将其与TT结合形成O-SP-TT结合疫苗,并用此结合疫苗免疫NIH小鼠,结果显示使用O-SP免疫后,小鼠血清中没有抗LPS抗体产生,而用L-SP-TT免疫后鼠血清中生了抗LPSIgG和IgM抗体,且IgG抗体水平高于IgM抗体;O-SP-TT免疫组等二次和第三次免疫后IgG我有显著的升高(P〈0.01),但第二次和第三次免疫后血清IgM 相似文献
7.
磷脂酰胆碱特异性磷脂酶C的研究 总被引:2,自引:0,他引:2
随着人们对信号转导认识的逐步加深,各种磷脂酶在信号通路中的作用也日渐受到重视,并日趋明了。其中磷脂酶A2(PLA2)、磷脂酰胆碱特异性磷脂酶D(PC-PLD)的基因已克隆,对磷脂酰肌醇特异性磷脂酶C(PI-PLC)也有较深了解,而对磷脂酰胆碱特异性磷... 相似文献
8.
广东眼镜王蛇毒酸性磷脂酶A2的分离纯化及抗血小板聚集作用 总被引:2,自引:0,他引:2
目的 研究眼镜王蛇毒中酸性磷酸脂酶A2对血小板的作用。方法 采用CM-Sephadesx C-25Sephadex G-75,DEAE-Sep「hadexA-25,Sephadex G-75多步柱层析法,聚丙烯酰胺产胶电泳,酸性磷脂酶A2酶活性测定;血小板聚集实验。累进要从粗中分离纯化出一酸性磷脂酶A2单体,分子量为14KD;等电点PI3.8;PLA2比值20μmol.mg^-1.min^-1;小 相似文献
9.
诱发肝癌过程中脂类和磷脂酶动态变化的相关性 总被引:1,自引:1,他引:0
研究了二乙基亚硝胺诱发大鼠肝癌过程1,2-甘油二酯(DAG),磷脂和磷脂酶的动态变化及其相互关系,高效薄层层析发现肝中DAG在第8周出现第一高峰,以后维持高于正常的水平,14周进一步增高,但以后下降,磷脂组分测定发现只有磷脂酰胆碱(PC)和肌醇磷脂类(PIs)在诱癌过程中降低,有可能成为DAG的来源,但PC可能是主要的,因PIs减少的量远小于DAG增加的量,进一步用酶偶联比色法测定PC专一性磷脂酶 相似文献
10.
百合的鳞茎中含有一种对木瓜蛋白酶有强抑制作用的巯基蛋白酶抑制剂.百合的鳞茎经浸取加热处理,木瓜蛋白酶偶联的Sepharose4B柱亲和层析和SephadexG-100分子筛层析,可获得在PAGE和SDS-PAGE均为单一蛋白带的百合巯基蛋白酶抑制剂(CPI).此CPI为单链蛋白,含有0.307%的中性糖;N端氨基酸为Ile;SDS-PAGE测得亚基分子量为12000;SephadexG-100测得分子量为12500.百合CPI在100℃内和pH2~12范围内非常稳定;对木瓜蛋白酶的抑制属竞争性抑制类型,其Ki值为1.15×10~(-9)mol/L,对木瓜蛋白酶的抑制摩尔比为8.5:1. 相似文献
11.
Murakami Y Kanzawa N Saito K Krawitz PM Mundlos S Robinson PN Karadimitris A Maeda Y Kinoshita T 《The Journal of biological chemistry》2012,287(9):6318-6325
Hyperphosphatasia mental retardation syndrome (HPMR), an autosomal recessive disease characterized by mental retardation and elevated serum alkaline phosphatase (ALP) levels, is caused by mutations in the coding region of the phosphatidylinositol glycan anchor biosynthesis, class V (PIGV) gene, the product of which is a mannosyltransferase essential for glycosylphosphatidylinositol (GPI) biosynthesis. Mutations found in four families caused amino acid substitutions A341E, A341V, Q256K, and H385P, which drastically decreased expression of the PIGV protein. Hyperphosphatasia resulted from secretion of ALP, a GPI-anchored protein normally expressed on the cell surface, into serum due to PIGV deficiency. In contrast, a previously reported PIGM deficiency, in which there is a defect in the transfer of the first mannose, does not result in hyperphosphatasia. To provide insights into the mechanism of ALP secretion in HPMR patients, we took advantage of CHO cell mutants that are defective in various steps of GPI biosynthesis. Secretion of ALP requires GPI transamidase, which in normal cells, cleaves the C-terminal GPI attachment signal peptide and replaces it with GPI. The GPI-anchored protein was secreted substantially into medium from PIGV-, PIGB-, and PIGF-deficient CHO cells, in which incomplete GPI bearing mannose was accumulated. In contrast, ALP was degraded in PIGL-, DPM2-, or PIGX-deficient CHO cells, in which incomplete shorter GPIs that lacked mannose were accumulated. Our results suggest that GPI transamidase recognizes incomplete GPI bearing mannose and cleaves a hydrophobic signal peptide, resulting in secretion of soluble ALP. These results explain the molecular mechanism of hyperphosphatasia in HPMR. 相似文献
12.
The copper-binding protein, ceruloplasmin, is both a serum component and a secretory product of Sertoli cells. Studies on serum ceruloplasmin have demonstrated it to be a ferroxidase that is essential for iron transport throughout the body. We report here that a glycosyl phosphatidylinositol (GPI)-anchored form of ceruloplasmin is expressed by Sertoli cells. Sertoli cell GPI-anchored proteins were selectively released by phosphatidylinositol-specific phospholipase C and were analyzed by Western blotting. A 135-kDa band was identified as ceruloplasmin by multiple antibody recognition and by amino acid sequence analysis. The presence of the GPI anchor on ceruloplasmin was confirmed by Triton X-114 phase partitioning experiments and by recognition with an antibody to the GPI anchor. GPI-anchored ceruloplasmin was enriched in detergent-insoluble glycolipid-enriched membrane microdomains (DIGs) of Sertoli cells. This is the first report of GPI-anchored ceruloplasmin in Sertoli cells and the first study of GPI-anchored ceruloplasmin in DIGs. We suggest that GPI-anchored ceruloplasmin may be the dominant form expressed by Sertoli cells and that Sertoli cell DIGs may play a role in iron metabolism within the seminiferous tubule. 相似文献
13.
Harada T Koyama I Sato K Komoda T 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2000,127(2):193-202
Serum alkaline phosphatase (ALP) is detected in soluble-form as a result of translocation from the membrane site by cleavage at the glycosyl-phosphatidylinositol moiety (GPI anchor). It is known that membrane-bound ALP (mALP) can be detected in serum in certain pathological and physiological conditions, and that it can be solubilized in vitro to soluble-ALP (sALP) by phosphatidylinositol-specific phospholipase C (PIPLC), phospholipase D, bile salt, detergent, etc. We observed a marked increase in ALP activity in the serum of rats given a benzimidazole derivative by gavage, and detected it as slow-migrating ALPs (SM-ALPs), which were mALP-like but resistant to PIPLC and n-butanol treatment on disc PAGE. On the other hand, ficin treatment made SM-ALPs shift to the sALP position. The molecular size of the SM-ALPs was smaller than that of sALP on sodium dodecyl sulphide-polyacrylamide slab-gel electrophoresis (SDS-PAGE), and immunoreactivity revealed the intestinal type. SM-ALPs were also detected in the duodenum and jejunum. The main sugar chain structure of SM-ALPs was the biantennary complex-type, which was coincided with intestinal sALP sugar chain. These results suggest that intestinal ALPs induced by the benzimidazole derivative were modified in their C-terminus or GPI anchor region and modification of this region may also participate in translocation into the bloodstream. 相似文献
14.
The purpose of this study was to examine the possibility of separation and quantification of serum alkaline phosphatase (ALP) isozymes in rats by wheatgerm lectin affinity electrophoresis. Cellulose acetate electrophoresis of the liver and bone ALPs without lectin results in overlapping bands, but in the presence of lectin, the mobility of the band of bone enzyme was retarded and well separated from the liver enzyme band. With this affinity electrophoretic method, we determined the serum ALP isozymes in fed and fasting rats grouped by age. As a result, the absolute activity of bone isozyme showed a downward trend with age in the fed and fasting rats. The serum ALP activity was steadily higher in fed rats than in fasting rats, and the increase was due to intestinal ALP isozyme. There was low activity bordering complete absence in liver isozyme under both nutritional conditions. The affinity electrophoretic method provided a rapid, reproducible, and relatively simple technique for further clinical characterization of ALP isozyme in the rat serum. 相似文献
15.
Martini CN Vaena de Avalos SG del Carmen Vila M 《Molecular and cellular biochemistry》2004,258(1-2):191-199
We have previously reported that ACTH activates a phospholipase C that hydrolyzes glycosylphosphatidylinositol (GPI), which would release inositolphosphoglycan (IPG) to the extracellular medium, and that an IPG purified from Trypanosoma cruzi is able to inhibit ACTH-mediated steroid production in adrenocortical cells. In the present paper, it was found that anti-inositolphosphoglycan antibodies (anti-CRD) increased ACTH-mediated corticosterone production, which indicates that an endogenous IPG is a physiological inhibitor of ACTH response. On the other hand, we investigated the release to the extracellular medium of the GPI-anchored enzyme, alkaline phosphatase, by ACTH. We found that: (a) the released enzyme appeared in the aqueous phase after Triton X-114 partitioning, consistent with loss of the GPI, (b) the phospholipase C inhibitor, U73122, impaired the release of the enzyme by the hormone and (c) two inhibitors of IPG uptake, inositol 2-monophosphate and 2 M NaCl, increased the amount of alkaline phosphatase in the extracellular medium. These results suggest that ACTH releases alkaline phosphatase by activation of a phospholipase C. Dibutyryladenosine-3',5'-cyclic monophosphate (db-cAMP) was able to increase the release of alkaline phosphatase from adrenocortical cells and this effect was inhibited by U73122, suggesting that cAMP is involved in the activation of phospholipase C. In addition, it was found that a pertussis-toxin sensitive G-protein is required for ACTH- and db-cAMP-mediated release of alkaline phosphatase and that incorporation of anti-Gi antibodies in adrenocortical cells inhibited the release of alkaline phosphatase by ACTH. Our results suggest that ACTH increases the release of alkaline phosphatase by activation of a phospholipase C through cAMP and Gi which would contribute to produce IPG It was also found that the two inhibitors of IPG uptake, inositol-2-monophosphate and 2 M NaCl, increased the amount of alkaline phosphatase in the extracellular medium of ACTH-treated cells more than in control cells, indicating that ACTH also stimulates the uptake of IPG These data support a role of GPI and the involvement of Gi in ACTH action. 相似文献
16.
The Streptomyces chromofuscus phospholipase D (PLD) cleavage of phosphatidylcholine in bilayers can be enhanced by the addition of the product phosphatidic acid (PA). Other anionic lipids such as phosphatidylinositol, oleic acid, or phosphatidylmethanol do not activate this PLD. This allosteric activation by PA could involve a conformational change in the enzyme that alters PLD binding to phospholipid surfaces. To test this, the binding of intact PLD and proteolytically cleaved isoforms to styrene divinylbenzene beads coated with a phospholipid monolayer and to unilamellar vesicles was examined. The results indicate that intact PLD has a very high affinity for PA bilayers at pH >/= 7 in the presence of EGTA that is weakened as Ca(2+) or Ba(2+) are added to the system. Proteolytically clipped PLD also binds tightly to PA in the absence of metal ions. However, the isolated catalytic fragment has a considerably weaker affinity for PA surfaces. In contrast to PA surfaces, all PLD forms exhibited very low affinity for PC interfaces with an increased binding when Ba(2+) was added. All PLD forms also bound tightly to other anionic phospholipid surfaces (e.g. phosphatidylserine, phosphatidylinositol, and phosphatidylmethanol). However, this binding was not modulated in the same way by divalent cations. Chemical cross-linking studies suggested that a major effect of PLD binding to PA.Ca(2+) surfaces is aggregation of the enzyme. These results indicate that PLD partitioning to phospholipid surfaces and kinetic activation are two separate events and suggest that the Ca(2+) modulation of PA.PLD binding involves protein aggregation that may be the critical interaction for activation. 相似文献
17.
18.
Kristiansen S Richter EA 《American journal of physiology. Endocrinology and metabolism》2002,283(2):E374-E382
We have previously developed a cell-free assay from rat skeletal muscle that displayed in vitro glucose transporter 4 (GLUT4) transfer from large to small membrane structures by the addition of a cytosolic protein fraction. By combining protein fractionation and the in vitro GLUT4 transfer assay, we have purified a glycosylphosphatidylinositol (GPI) phospholipase D (PLD) that induces transfer of GLUT4 from small to large membranes. The in vitro GLUT4 transfer was activated and inhibited by suramin and 1,10-phenanthroline (an activator and an inhibitor of GPI-PLD activity, respectively). Furthermore, upon purification of the GLUT4 transporter protein, the protein displayed an elution profile in which the molecular mass was related to the charge, suggesting the presence or absence of phosphate. Second, by photoaffinity labeling of the purified GLUT4 with 3-(trifluoromethyl)-3-(m-[(125)I]iodopenyl)diazirine, both labeled phosphatidylethanolamine and fatty acids (constituents of a GPI link) were recovered. Third, by using phase transition of Triton X-114, the purified GLUT4 was found to be partly detergent resistant, which is a known characteristic of GPI-linked proteins. Fourth, the purified GLUT4 protein was recognized by an antibody raised specifically against GPI links. In conclusion, GLUT4-containing vesicles may be released from a membrane compartment by action of a GPI-PLD. 相似文献
19.
S Das A Traynor-Kaplan D S Reiner T C Meng F D Gillin 《The Journal of biological chemistry》1991,266(31):21318-21325
Since Giardia lamblia trophozoites are exposed to high concentrations of fatty acids in their human small intestinal milieu, we determined the pattern of incorporation of [3H]palmitic acid and myristic acid into G. lamblia proteins. The pattern of fatty acylation was unusually simple since greater than 90% of the Giardia protein biosynthetically labeled with either [3H]palmitate or myristate migrated at approximately 49 kDa (GP49) in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis during both growth and differentiation. GP49, which partitions into the Triton X-114 detergent phase, is localized on the cell surface since it is 125I-surface-labeled. GP49 was also biosynthetically labeled with [14C]ethanolamine and [3H]myoinositol, suggesting that it has a glycosylphosphatidylinositol (GPI) anchor. Moreover, phospholipase A2 (PLA2) or mild alkaline treatment released free fatty acids, indicating a diacylglycerol moiety with ester linkages. Finally, a 3H- and 14C-labeled species was released by nitrous acid deamination from [14C]palmitate- and [3H]myoinositol-labeled GP49. The GPI anchor of GP49 is unusual, however, because purified GP49 was cleaved by Bacillus cereus phosphatidylinositol (PI)-specific PLC, but not by Staphylococcus aureus PI-PLC, or plasma PLD, and did not react with antibody against the variant surface glycoprotein cross-reactive determinant. Moreover, the double-labeled deaminated GP49 anchor migrated faster than authentic PI in TLC and produced [3H]glycerophosphoinositol after deacylation. In contrast to the variable cysteine-rich G. lamblia surface antigens described previously, GP49 was identified in Western blots of every isolate tested, as well as in subclones of a single isolate which differ in expression of a major cysteine-rich 85/66-kDa surface antigen, which does not appear to be GPI-anchored. These observations suggest that GP49, the first common surface antigen to be described in G. lamblia, may play an important role in the interaction of this parasite with its environment. 相似文献
20.
Khodagholi F Yazdanparast R Sadeghirizi A 《Journal of biomolecular structure & dynamics》2007,25(2):189-194
Regarding the world wide success of artificial chaperone-assisted protein refolding technique and based on its well worked-out mechanism, it is anticipated that the lipid moieties of glycosylphosphatidylinositol (GPI) group, which is present in some membrane proteins, might interfere with the capturing step of the technique. To find an answer, we evaluated the chemical denaturation and also the refolding behavior of insoluble and soluble alkaline phosohatase (ALP), with or without GPI group, respectively. The results indicated that the presence of GPI in the enzyme increased the stability of the protein against chemical denaturation while it decreased its refolding yield by the artificial chaperone refolding technique. The lower refolding yield, compared to soluble ALP (sALP), might be due to a less efficient stripping step caused by new interactions imparted to the refolding elements of the system especially those among the hydrophobic tails of GPI and the capturing agent of the technique. These new interactions will interrupt the kinetics of detergent stripping from the captured molecules by the stripping agent (i.e., cyclodextrins). This situation will lead to higher intermolecular hydrophobic interactions among the refolding protein intermediates leading to their higher misfolding and aggregation. 相似文献