人血清中小分子蛋白质的电洗脱回收

[目的]建立一种快速简便的回收分子量﹤10000 Da低丰度蛋白质的电洗脱方法。[方法]采用改进后的聚丙烯酰胺凝胶电泳和电洗脱方法分离纯化小分子标志蛋白,利用SELDI技术检测回收后的标志蛋白。[结果]SELDI技术检测发现,改进后的电泳和电洗脱方法能有效分离并回收10000 Da以下的低丰度蛋白质。[结论]改进后的电洗脱方法,不仅快速简便,而且还有利于蛋白质的后续鉴定。     

乙腈沉淀法去除人血清中高丰度蛋白的探索性研究

目的:探索乙腈沉淀法去除人血清中高丰度蛋白的效果。方法:应用不同体积的乙腈对血清样品进行处理,Tric ine-SDS-PAGE电泳检测去除高丰度蛋白的效果。结果:乙腈沉淀法能在去除血清高中丰度蛋白的同时收获更多的10 kD以下的小分子量蛋白,2倍体积乙腈结合一定浓度的TFA能有效地去除血清高中丰度蛋白。结论:乙腈沉淀法能去除血清中高丰度蛋白,是一种分离血清部分小分子量蛋白质简便、经济、有效的前期处理方法。     

两种去除血清高丰度蛋白方法的比较

目的比较乙腈沉淀法和MERCK公司的血清白蛋白和IgG去除试剂盒去除人血清中高丰度蛋白的效果。方法应用乙腈沉淀法和MERCK公司的血清白蛋白和IgG去除试剂盒对血清样品进行处理,Tricine-SDS-PAGE电泳和SELDI-TOF-MS检测去除高丰度蛋白的效果。结果乙腈沉淀法能在去除血清高中丰度蛋白的同时收获更多的10kD以下的小分子量蛋白,MERCK公司的试剂盒也能有效地去除血清高中丰度蛋白,特异性较好。结论2种方法均能去除血清中高丰度蛋白,需根据研究目的选择适合的方法。     

人外周静脉血血清蛋白质双向凝胶电泳条件优化

目的优化人外周静脉血血清蛋白质的双向凝胶电泳条件。方法血清总蛋白经去除白蛋白、IgG和丙酮沉淀去盐分处理后,固相pH梯度等电聚焦(IEF)和十二烷基硫酸钠-聚丙烯酰胺凝胶(SDS-PAGE)进行蛋白质电泳,对蛋白质的上样量、固相pH梯度(IPG)胶条的pH值范围、凝胶浓度、水化上样液体积和IEF程序及其参数等条件进行优化。结果将50～100μg蛋白质溶解于终体积为350μl水化上样液,应用pH4～7的IPG胶条和浓度12%的SDS-PAGE凝胶进行双向电泳,经优化电泳电流、时间等参数后,建立了合适的双向凝胶电泳条件,得到比较理想的蛋白质二维图谱。结论优化了血清蛋白质双向凝胶电泳条件,为进一步开展血清差异蛋白质组学研究奠定基础。     

乙腈、乙醇、色谱柱去除血清高丰度蛋白方法的比较

目的比较乙腈沉淀法、乙醇沉淀法和多重亲和去除色谱柱Human 14法去除人血清中高丰度蛋白的效果。方法应用乙腈沉淀法、乙醇沉淀法和多重亲和去除色谱柱Human 14法对血清样品进行处理,Bis-Tris Mini Gels电泳和双向凝胶电泳检测去除高丰度蛋白的效果。结果从一维胶电泳图灰度值分析,血清经过乙腈法、色谱柱法、乙醇法处理后,白蛋白条带灰度值由原血清的19分别变成157.2,40.8和8.2;从2-DE电泳图分析看出,多重亲和去除色谱柱Human 14法处理后的蛋白点数显著增多,比原血清增加了137个;乙腈法和乙醇法处理后虽然蛋白点减少了,却有低丰度蛋白质点显现出来。结论乙腈沉淀法能在去除血清中高丰度蛋白的同时收获更多的10kD以下的小分子量蛋白;乙醇沉淀法虽然能去除部分高丰度蛋白,但小分子量蛋白的收获量较少;而多重亲和去除色谱柱Human 14法不仅能有效地去除血清中高丰度蛋白的干扰,且保留了大量的小分子量蛋白。     

临床实验室检查结果解读 血清特种蛋白的种类和临床意义

血清中的蛋白质,用一般电泳的方法可以分离为白蛋白、αl、α2、β、γ球蛋白等几条带。利用一些新技术,可以将这几条带中的成分继续分离为各种异质的蛋白质。例如,γ球蛋白可分离为免疫球蛋白(G、A、M、E、D)、补体、血型球蛋白、冷     

食品中蛋白质含量测定方法的探讨

目的:建立一种纳氏比色法测定食品中蛋白含量的检测方法。方法:样品经前处理后,在420nm波长处用纳氏比色法进行蛋白质含量测定。结果:本方法在氮含量为0.1～2.0mg/L范围内线性良好。样品蛋白质含量在2.80%～66.3%之间的相对标准偏差为0.96%～3.13%。该法与凯氏定氮法对不同样品进行蛋白质含量检测,无显著统计学差异。结论:该方法操作简便、快速、准确。     

高血压脑出血肝阳化风证与肝阳上亢证患者血清蛋白质组学研究

目的初步建立高血压脑出血肝阳化风证、高血压肝阳上亢证及健康对照的血清蛋白质组表达图谱,初步鉴定其差异表达的蛋白质。方法各组血清均以试剂盒去除白蛋白及IgG,以BCA法测血清蛋白浓度。以双向凝胶电泳分离各组血清蛋白质,考马斯亮蓝染色,扫描凝胶成像,以PDQuest软件对各组血清蛋白质组表达图谱进行差异分析,选定差异点进行MALDI-TOF质谱分析。Mascot软件搜索MSDB和SWISS-PROT数据库鉴定蛋白质。结果⑴初步建立三组血清蛋白质表达图谱。⑵8个蛋白质点在三组血清中呈现差异表达,初步鉴定其中的5个蛋白质:血清淀粉样前体蛋白,铜蓝蛋白,维生素D结合蛋白,载脂蛋白C-III和转铁蛋白。结论得到鉴定的5个蛋白质可能与高血压脑出血肝阳化风证发病过程相关。     

血清中小分子蛋白电泳的影响因素

SDS-聚丙烯酰胺凝胶电泳是一种有效分离血清中小分子蛋白的方法,但电泳的分辨率与电泳的凝胶浓度、缓冲液的选择、样品的制备和染色等诸多因素密切相关.现就这些影响因素作一简要分析.     

纸上电泳法的简单装置及一些正常人及病人的血清蛋白质电泳数值

本研究报告纸上电泳法的简单装置,并找出了使用此装置的最适宜情况。本文略述光密度计及电压稳定器的自制方法。 在研究中证明用偶氮胭脂红B染色,用光密度计法定量是较简便的血清蛋白质定量方法,所得的结果与Kjeldahl微量定氮法所得者符合,在球蛋白部分无需乘以任何校正系数。本法的重复性亦曾加以测定。 我们测定了60个正常人的血清蛋白质电泳数值和一些病人血清蛋白质电泳数值。     

SELDI技术检测反义寡核苷酸作用前后肝癌细胞培养液的差异蛋白

目的利用针对人端粒酶RNA(hTR)的反义寡核苷酸(ASODN)和正义寡核苷酸(NODN)作用于人肝癌细胞SMMC-7721,比较ASODN和NODN作用后细胞培养液蛋白的变化。方法利用SELDI技术检测差异蛋白的表达变化。对照组为未加任何处理因素的SMMC-7721细胞。结果SELDI技术检测发现,ASODN作用组有40个差异蛋白分子低表达,3个差异蛋白分子高表达。NODN作用组有37个差异蛋白分子低表达,6个差异蛋白分子高表达。所有差异蛋白分子量均小于10 000 Da。ASODN作用组中有3个低表达蛋白在NODN作用组高表达,分子量分别为3 011.99 Da、3 048.28 Da和3 248.75 Da。结论ASODN和NODN作用SMMC-7721细胞后细胞培养液中所表达的差异蛋白十分相似。     

Modification of serum proteins in guinea pigs immunized and challenged with toluene diisocyanate

OBJECTIVES: Guinea pigs were used to determine whether immunization and challenge by toluene diisocyanate (TDI) induce changes in the serum protein concentrations of the "acute-phase response" and whether TDI can form adducts with serum proteins. METHODS: Guinea pigs were immunized by weekly intradermal injections of TDI and challenged with TDI 7 days after the 3rd injection. The animals were killed 6 hours after the challenge, and serum was analyzed for protein characterization by gel electrophoresis and for specific antibodies to TDI by enzyme-linked immunosorbent assay (ELISA) and Western blotting. RESULTS: The total serum protein concentration of the immunized TDI-challenged guinea pigs increased in comparison with that of nonimmunized animals [75 (SE 0.7) versus 47.4 (SE 2.3) mg/ml; ]. Albumin and alpha, and alpha2 globulins increased significantly [respectively: 65.8 (SE 0.2)%, 2.1 (SE 0.1)% and 7.2 (SE 0.1)% versus 59 (SE 1.3)%, 1.3 (SE 0.1)% and 3.7 (SE 0.1)%], whereas beta1 and beta2 globulins decreased in the immunized TDI-challenged guinea pigs [7.8 (SE 0.2)% and 0.8 (SE 0.2)% versus 15.8 (SE 0.7)% and 4.8 (SE 0.2)%]. The gamma globulin concentrations did not change significantly. In the immunized TDI-challenged animals, albumin was modified by TDI and ran faster on agarose gel electrophoresis than did albumin from nonimmunized guinea pigs. In the ELISA, only immunized animals had high titers of TDI-specific antibodies (IgG and IgG1); by blotting, the antibodies reacted against TDI, the TDI-BSA-conjugate and several TDI-conjugated guinea pig serum proteins, but they did not react against any native or denaturated serum protein when unconjugated with TDI. CONCLUSIONS: These findings indicate that, in guinea pigs, immunization and challenge with TDI induces changes in serum proteins of the "acute phase response" and TDI is adducted to serum proteins with different molecular weights (eg, albumin).     

Regulation of the formation of stable adducts between acetaldehyde and blood proteins

Recent reports have described increased levels of a fast-moving hemoglobin (Hb) fraction in alcoholic patients and formation in vitro of stable adducts between acetaldehyde and Hb as well as between acetaldehyde and albumin. In the present study, we have found that factors other than acetaldehyde concentration can influence the rate of stable adduct formation. HbAo was purified and its 2,3-diPglycerate removed by dialysis. Under anaerobic condition and at 37 degrees, acetaldehyde (5 microM) reacted with deoxyHbAo to form 0.26 +/- 0.02 (+/- SEM) nmol of stable adduct/149 nmol Hb in 2 hr. By comparison, acetaldehyde reacted more slowly with oxyHbAo and carbonylHbAo; the rates were 0.21 +/- 0.01 (p less than 0.001) and 0.18 +/- nmol/149 nmol Hb/2 hr (p less than 0.001), respectively. Pyridoxal 5'-phosphate (50-500 microM), under anaerobic condition, inhibited by 36-56 percent the irreversible binding of acetaldehyde to deoxyHbAo. Ascorbic acid (2.5-10 mM) increased by 31-46 percent (p less than 0.001) the irreversible binding of acetaldehyde to human serum albumin and by 8-10 percent (p less than 0.05) the irreversible reaction of acetaldehyde with serum proteins. We conclude that the nonenzymatic binding of acetaldehyde to Hb, human serum albumin and serum proteins is influenced by factors other than acetaldehyde concentration. These factors include oxygen tension, pyridoxal 5'-phosphate and ascorbic acid. Among these factors, oxygen tension may be the most important in vivo.     

Cerebrospinal fluid changes in tetanus: raised proteins and immunoglobulins in patients with severe disease

34 patients with tetanus were studied; all had normal serum proteins and albumin. Only 3 samples of cerebrospinal fluid (CSF) contained cells (a few polymorphonuclear leucocytes). Protein levels in the CSF were elevated in 26 patients (76.5%); 11 cases with clinically mild tetanus had a mean CSF protein of 400 +/- 250 mg/litre (normal 100-400 mg/litre), while the CSF protein levels of 10 patients who died (mean 1596 +/- 985 mg/litre; P less than 0.005) and those of 4 others with severe disease who absconded were much higher (mean 1220 +/- 562 mg/litre; P less than 0.005). As a group, 19 patients with severe disease including the 10 fatal cases also had significantly higher mean CSF protein values (1582 +/- 938 mg/litre; P less than 0.005) than did the mild cases. Immunochemical analysis of the proteins using a radial immunodiffusion assay showed that these proteins were immunoglobulin G (IgG). Polyacrylamide gel electrophoresis demonstrated an oligoclonal gammaglobulin pattern, suggesting intrathecal IgG synthesis in these patients. These studies suggest that measurement of CSF proteins may supplement clinical evaluation in tetanus and that there may be a local immune stimulus (perhaps a tetanus antigen or to some other mitogen) in the central nervous system.     

Spectrum of antimicrobial activity and user acceptability of the hand disinfectant agent Sterillium Gel

The antimicrobial efficacy of alcohol-based hand gels has been shown to be significantly less than liquid hand rubs probably because of a lower concentration of alcohol. Sterillium Gel is the first hand gel with 85% ethanol. Its antimicrobial efficacy and user acceptability was studied. Bactericidal activity was tested according to prEN 12054 against Staphylococcus aureus, Enterococcus hirae, Pseudomonas aeruginosa and Escherichia coli (suspension test) and EN 1500 (15 volunteers; four replicates), fungicidal activity according to EN 1275 against Candida albicans and spores of Aspergillus niger (suspension test) and tuberculocidal activity against Mycobacterium terrae using the DGHM suspension test. Virucidal activity was determined in suspension tests based on reduction of infectivity with and without interfering substances (10% fetal calf serum; 0.3% erythrocytes and 0.3% bovine serum albumin). Ninety-six healthcare workers in hospitals in France and the UK used the gel for four weeks and assessed it by filling out a questionnaire. The gel was bactericidal (a reduction factor of > 10(5)-fold), tuberculocidal (reduction factor > 10(5)) and fungicidal (reduction factor > 10(4)) in 30 s. Irrespective of interfering substances the gel inactivated orthopoxvirus and herpes simplex virus type 1 and 2 in 15 s, adenovirus in 2 min, poliovirus in 3 min and papovavirus in 15 min by a factor of > 10(4)-fold. Rotavirus and human immunodeficiency virus were inactivated in 30 s (without interfering substances). Under practical use conditions it was as effective in 30 s as the reference alcohol in 60 s. Most users described the tackiness, aggregation, skin feeling after use and smell as positive or acceptable. A total of 65.6% assessed the new gel to be better than a comparator irrespective of its type (gel or liquid). Overall Sterillium Gel had a unique spectrum of antimicrobial activity. It is probably the first alcohol-based hand gel to pass EN 1500 in 30 s. Due to the excellent acceptance by healthcare workers it may significantly improve compliance for hand hygiene and thereby help to reduce the incidence of nosocomial infection.     

Immunogenicity in mice of pneumococcal glycoconjugate vaccines using pneumococcal protein carriers

Antibody response and protective immunity were evaluated in mice immunized with pneumococcal glycoconjugate vaccines using two pneumococcal protein carriers. Mice injected with type 9V polysaccharide (PS) conjugated to inactivated pneumolysin (Ply) or autolysin (Aly) produced high levels of IgG and IgM antibodies to both the PS and the protein carrier. Higher PS antibody titers to the pneumococcal PS conjugates were measured by ELISA using PS-Ply or PS-tetanus toxoid (TT) conjugate as a coating antigen compared with PS mixed with methylated human serum albumin. Type 9V PS (10 microg) inhibited most of the 9V IgM and IgG antibody binding to the 9V-TT coated plate. In contrast, absorption with 19F PS did not inhibit 9V antibody binding. The avidity index of IgG antibodies in the 9V PS-Ply serum was 55.5 +/- 0.9, compared with 47.8 +/- 1.4 for 9V PS-Aly serum. Thus, high avidity of serum antibodies in conjugate-immunized mice can provide more effective functional activity for protection against pneumococcal infection. Mice immunized with these glycoconjugates exhibited rapid bacterial clearance from blood and provided cross-protection against challenge with heterologous serotypes of virulent pneumococci. These results reveal that conjugates using pneumococcal protein carriers can induce opsonophagocytic activity to destroy homologous and heterologous pneumococci, indicating that such conjugates can confer broader protective immunity than conjugates using non-pneumococcal proteins.     

输卵管妊娠血β-hCG值与滋养细胞浸润输卵管肌层的关系

目的:探讨输卵管妊娠患者血β-hCG水平与滋养细胞浸润输卵管肌层的关系。方法:测定106例接受输卵管切除术的输卵管妊娠患者术前血β-hCG值。根据术后病理检查结果将患者分为A组(有滋养细胞浸润输卵管肌层)62例和B组(无滋养细胞浸润输卵管肌层)44例。计算病例的血β-hCG值百分位数,确定血β-hCG的阳性判断界限值。结果:106例患者血β-hCG值为(4890±763)IU/L。A、B两组间血β-hCG值的差异有显著性(P<0.01)。取3000IU/L为判断有无滋养细胞浸润输卵管肌层的血β-hCG界限值,诊断的准确率为93.40%,敏感度96.77%,特异度88.64%。结论:输卵管妊娠患者血β-hCG值水平与滋养细胞浸润输卵管肌层有明显的关系,血β-hCG值≥3000IU/L时,将发生滋养细胞浸润输卵管肌层。     

金黄色葡萄球菌的蛋白组学研究

目的 通过表面增强激光解析电离飞行时间质谱(SELDI)技术比较甲氧西林敏感金黄色葡萄球菌(MSSA)与耐甲氧西林金黄色葡萄球菌(MRSA)的蛋白质组学特点及差异.方法 首先采用K-B纸片扩散法进行药敏试验,然后SPA法分别鉴定出MSSA与MRSA同源性菌株,通过利用SELDI技术对其蛋白进行检测,并采用Biomarker Wizard软件分析其蛋白质谱特点.结果 菌株型为t030的同源性菌株17085及16982有3个蛋白质表达有差异,分子量分别为3057、5867、11 511Da;菌株型为t037的同源性菌株18565及68也仅有3个蛋白表达有差异,分子量分别为4304、7492、8354 Da;非同源性菌株18565、16982(均为MRSA)有6个蛋白质表达有差异,分子量分别为3057、5521、6416、7340、8877、11 459 Da;非同源性菌株17085、68(均为MSSA)有7个蛋白质表达有差异,分别为3100、5520、6416、6888、8079、8876、12 366Da.结论 同源性菌株SELDI蛋白质谱具有高度相似性,非同源的MRSA或MSSA蛋白质谱表达均有较大差异.