检测脑膜炎球菌菌苗中内毒素方法的研究

作者应用聚丙烯酰胺凝胶电泳结合银染(SDS—PAGE银染)和2—酮基—3—脱氧辛糖(KDO)方法来测定脑膜炎球菌外膜蛋白和多糖菌苗中的脂多糖(LPS)。这二种方法能比较真实地测定出存在于蛋白或多糖制品中的LPS实际含量。其结果和家兔热原质试验一致。KDO法并能定量测定蛋白样品中的LPS含量。     

Salivary Amylase Induction by Tannin-Enriched Diets as a Possible Countermeasure Against Tannins

Tannins are characterized by protein-binding affinity. They have astringent/bitter properties that act as deterrents, affecting diet selection. Two groups of salivary proteins, proline-rich proteins and histatins, are effective precipitators of tannin, decreasing levels of available tannins. The possibility of other salivary proteins having a co-adjuvant role on host defense mechanisms against tannins is unknown. In this work, we characterized and compared the protein profile of mice whole saliva from animals fed on three experimental diets: tannin-free diet, diet with the incorporation of 5% hydrolyzable tannins (tannic acid), or diet with 5% condensed tannins (quebracho). Protein analysis was performed by one-dimensional gel electrophoresis combined with Matrix-Assisted Laser Desorption Ionization-Time of Flight mass spectrometry to allow the dynamic study of interactions between diet and saliva. Since abundant salivary proteins obscure the purification and identification of medium and low expressed salivary proteins, we used centrifugation to obtain saliva samples free from proteins that precipitate after tannin binding. Data from Peptide Mass Fingerprinting allowed us to identify ten different proteins, some of them showing more than one isoform. Tannin-enriched diets were observed to change the salivary protein profile. One isoform of α-amylase was overexpressed with both types of tannins. Aldehyde reductase was only identified in saliva of the quebracho group. Additionally, a hypertrophy of parotid salivary gland acini was observed by histology, along with a decrease in body mass in the first 4 days of the experimental period. G. da Costa and E. Lamy have contributed equally to this work.     

流感疫苗血凝素含量测定替代方法的建立及验证

目的建立测定流感疫苗血凝素(Haemagglutinin,HA)含量的替代方法 ,并进行验证,以解决大流行流感发生初期疫苗原液中HA定量的难题。方法优化糖苷酶处理条件,将流感疫苗原液经糖苷酶处理后,以还原SDS-PAGE方法分离样品,以灰度扫描法确定HA蛋白百分比,结合样品总蛋白数值,计算样品中HA含量。以该方法和传统的单向免疫扩散(SRID)法分别测定7批流感疫苗原液中HA含量,并进行比较。结果优化的糖苷酶处理条件为:总蛋白400μg/ml,PNGaseF加入比例为1:50。样品经糖苷酶处理后,以还原SDS-PAGE法可以清晰区分各蛋白条带,经测序后确定HA两个亚基,与预期相对分子质量一致。该方法测定7批流感疫苗原液中HA含量的结果与SRID法测定结果的符合率在87.90%~122.20%之间。结论初步建立了测定流感疫苗中HA含量的替代方法 ,在WHO参考品供应不到位时,可用于大流感发生时疫苗原液中HA之定量。     

The effect of enzyme systems and processing on the hydrolysis of peanut (Arachis hypogaea L.) protein

In vitro protein digestion studies were carried out on raw and roasted peanut flour as the starting material in the production of peanut protein hydrolysate. Peanut flour was hydrolyzed with alcalase and alternately in a sequential digestion with pepsin-pancreatin, both for up to 24 h. The degree of hydrolysis (DH) at different times of hydrolysis was determined using the trinitrobenzenesulfonic acid (TNBS) method. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to indicate destruction of native protein units in the enzymatic digests.Hydrolysis with alcalase was very rapid for the first 6 h after which a plateau was reached, whereas that with pepsin–pancreatin was more gradual reaching a plateau after 12 h of hydrolysis. Raw peanut hydrolyzed with alcalase and pepsin–pancreatin had 23% and 21% DH after 24 h respectively, whilst roasted peanut hydrolyzed with alcalase had 21% DH, with the pepsin–pancreatin hydrolysate recording the highest value of 25% after 24 h of hydrolysis.SDS-PAGE results showed that raw peanut samples behaved differently from the roasted samples; increasing hydrolysis time reduced larger peanut protein subunits, with only peptides of <20 kDa visible after hydrolysis for raw peanut, and virtually no distinct visible bands for the roasted peanut after 3 h of hydrolysis.     

Characterisation of the S-Carboxymethyl Extract of Wool Using Small Format Alkaline Urea-PAGE/SDS-PAGE Followed by Silver Staining

Abstract

Alkaline urea-PAGE/SDS-PAGE, when used in a novel format which was significantly smaller than that employed by earlier workers, and when followed by silver staining, resulted in the detection of excellently resolved protein components from S-carboxymethyl reductive extracts of very small wool samples, even samples as small as an individual wool fibre. The silver stained two-dimensional gel patterns exhibited significant improvements compared to the fluorograph gel patterns of earlier workers based on the presence of radiolabel incorporated within the S-carboxymethyl moiety. The silver staining resulted in the visualisation of numerous protein components, and in the region of the gel pattern expected to contain the high-sulfur protein fraction, there appeared more major components than the number of high-sulfur protein components usually displayed in fluorograph patterns. The relative amount of the high-sulfur protein fraction in the final silver stained gel pattern could be boosted, if desired, if the gel was loaded not with the whole wool extract but with the filtrate resulting from the passage of the whole extract through a centrifugal ultrafilter.     

唐菖蒲两变异株的同功酶及SDS-聚丙烯酰胺凝胶电泳的比较分析

为探讨电子束对唐菖蒲诱变的可行性及不同剂量电子束对其花性状的影响，用不同剂量电子束辐照唐菖蒲“江山美人”球茎，在40Gy和160Gy处理组分别得到了1株花色和花序变异株(M1’和M2’)。对这两变异株和对照以及其相应辐照剂量（40Gy和160Gy）处理组进行了研究。M1代植株叶片的过氧化物酶、过氧化氢酶、淀粉酶和酯酶4种同功酶电泳结果表明，40Gy和160Gy处理组的酶带与对照组的相同，变异株与对照组相比，酶带有所增减。基于同功酶谱带带型，使用SPSS11.5进行了聚类分析并得到聚类树状图。图中显示，供试材料被分成了3个组：对照组（包括40Gy和160Gy处理组与对照），M1’组和M2’组。SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析表明，蛋白表达明显被电子束辐照所抑制，但在这两个变异株中观测到3条特异表达的蛋白条带，分子量分别为96kDa、115.4kDa和137.2kDa，这些特异表达的蛋白可能与花色与花序的调控有关。由此表明，电子束辐照诱导花色与花形突变体是1种有效的途径。     

部分小麦品种的高相对分子质量谷蛋白亚基组成分析

利用电泳技术分析了94个国产小麦样品的高相对分子质量谷蛋白亚基组成,结果表明:这些小麦品种大部分具有1A上的优质亚基1,1B上的优质亚基7 8/17 18,1D上的5 10,个别品种还同时具有1A、1B、1D上的优质亚基;我国的小麦品种高相对分子质量谷蛋白亚基组成类型较丰富,共检测到16种亚基和28种亚基组合类型,品质评分4~10分,平均为6.9分;在小麦品种中,含有优质亚基5 10、17 18或2*,分别有44、5和13个品种,可供优质小麦育种应用.     

大豆蛋白质11S和7S组分及其亚基分析方法的研究述评

对大豆蛋白质组分及其亚基的分析方法进行了评述.主要内容为:利用超速离心技术把大豆蛋白质组分分为4种,以沉降系数分别表示为2S、7S、11S和15S,其中7S和11S是主要组分;利用碱溶、酸沉(冷沉)和缓冲液中沉淀的方法分别分离提取7S和11S,经凝胶过滤或离子交换柱层析提纯;利用碱溶、酸沉和离心分离方法分离提取大豆分离蛋白;利用D isc-PAGE技术初步分析7S和11S组分的个别亚基,利用SDS-PAGE技术测定7S和11S组分及其亚基的相对分子质量和相对含量;以相对分子质量为标准,在SDS-PAGE谱带中划分7S和11S组分的亚基组并测定其相对含量,然后计算7S和11S组分的相对含量和11S/7S比值.上述方法各有其优缺点和适用情况.在讨论的基础上提出食品科学与遗传育种科学要密切结合,培育含有不同蛋白质组分、不同亚基和组分比值的专用大豆品种,既可以深入研究亚基的功能特性,又能开发出新的大豆蛋白制品.     

缢蛏过敏原的分离、鉴定与纯化

以磷酸盐缓冲液提取蛋白,通过聚丙烯酰胺凝胶电泳(SDS-PAGE)对其抗原成分进行分析,选用缢蛏过敏患者的阳性血清进行免疫印迹,鉴定出主要与次要过敏原.用高压液相色谱(HPLC)纯化主要过敏原蛋白,鉴定其免疫活性,结果表明,缢蛏粗提液SDS-PAGE显示有18条蛋白条带,含量高的蛋白有6条,其分子量分别为110kD、55kD、42kD、38kD、35kD和28kD,其中以35kD处最为富集.经Western-Blotting鉴定,58kD蛋白为缢蛏的主要过敏原,38kD、28kD和12kD蛋白为缢蛏的次要过敏原.HPLC体积排阻纯化后得9个峰,以SDS-PAGE检测,58kD的蛋白位于第4峰,其中有一条明显的蛋白条带.将该蛋白条带用HPLC反相纯化,得到两个峰.经Western-Blotting鉴定,58kD的主要过敏原位于反相纯化的第2峰.     

Microstructure and protein digestibility of beef: The effect of cooking conditions as used in stews and curries

Beef meat was cooked at 373 K for 10 and 30 min to investigate the effect of the cooking conditions generally used during beef stew and curry preparation on protein digestibility. The cooked meats, along with a raw control, were digested using an in vitro digestion model to simulate gastric and small-intestinal conditions. Samples taken at different digestion times were analyzed using SDS-PAGE, RP-HPLC, ninhydrin assays for amino N and transmission electron microscopy. Simulated gastric conditions quickly led to the loss of basic sarcomere structure in raw meat myofibrils whereas the sarcomere structure of the compact cooked meat myofibrils remained intact after 30 min of gastric digestion. Prolonged cooking of meat (30 min) resulted in incomplete digestion of small MW (<10 kDa) peptides, as observed from SDS-PAGE. This agreed with the amount of ninhydrin-reactive amino N released during digestion, which decreased with an increase in cooking time. The RP-HPLC peak areas of the major identified amino acids (tryptophan, tyrosine and phenylalanine) also decreased with an increase in cooking time. This suggested the formation of “limit peptides” during prolonged cooking of beef, which were not further broken down into free amino acids by digestive enzymes and therefore might not be bioavailable.