刀额新对虾变应原的分离、鉴定与纯化

目的通过对我国常见的刀额新对虾变应原蛋白进行分离、鉴定与纯化，以提供虾特异性变应原用于食物变态反应疾病的诊断和治疗。方法通过十二烷基硫酸钠一聚丙烯酰胺凝胶电泳（SDS—PAGE）分离刀额新对虾的蛋白质组分并测定其分子量，收集过敏病人血清，采用免疫印迹（Western—blotting）法鉴定其变应原成分，通过离子交换层析对刀额新对虾变应原进行初步纯化，通过疏水层析和凝胶过滤层析进一步对变应原纯化。结果刀额新对虾有17条蛋白带，其中主要条带有10条，68和36kD为可与虾过敏性病人血清IgE结合的特异性变应原；通过各种层析方法纯化出刀额新对虾68kD变应原，纯度为96％。结论对刀额新对虾变应原进行分离、鉴定和纯化，得到高纯度的68kD刀额新对虾变应原。为临床虾变态反应疾病的诊断和治疗奠定基础。     

鲤鱼主要变应原的分离、鉴定与纯化

目的 对常见的食物过敏原鲤鱼进行特异性抗原成分的分离、鉴定和纯化，为临床诊断及治疗鲤鱼过敏症提供理论依据。方法 通过磷酸盐缓冲溶液进行提取、用十二烷基硫酸钠．聚丙烯胺凝胶电泳（SDS-PAGE）分离出多种蛋白组分并测定其分子量；采用蛋白印迹技术（Western-blotting）鉴定其变应原并通过DE52离子交换层析对鲤鱼变应原进行初步纯化，进一步经凝胶过滤层析对变应原混合组分进行纯化。结果 SDS-PAGE分离出14种蛋白组分，其中主带有9条，分子量分别为5，46，36，34，26，25，23，20，16kDa。Western—blitting结果显示，鲤鱼的主要变应原及次要变应原的分子量是42，36kDa的变应原组分。结论 鲤鱼主要特异性变应原为42和36kDa的蛋白质，为临床诊断和治疗鱼类过敏性疾病提供标准化的廊原尊定了基础．     

鸡蛋过敏原分离、鉴定与纯化

目的 对鸡蛋中主要过敏原进行分离、鉴定与纯化.方法 分别提取鸡蛋的蛋清与蛋黄的蛋白,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离鸡蛋的蛋白质组份,采用免疫印迹(Western-blotting,wB)方法鉴定过敏原,通过离子交换层析对鸡蛋主要过敏原进行初步纯化.结果 WB检测显示,蛋黄与鸡蛋过敏患者的阳性混合血清没有反应;蛋清中有4条蛋白带起反应,分子量分别为83,71,38,13 kD,尤以13 kD的蛋白质最为明显,离子交换层析可初步纯化出13 kD的过敏原蛋白.结论 鸡蛋的主要过敏原为13 kD的蛋白质,为临床诊断和治疗鸡蛋过敏性疾病提供标准化的过敏原提供了基础依据.     

幽门螺杆菌HspA乳酸菌疫苗构建及免疫反应性鉴定

目的 探讨幽门螺杆菌(Hp)热休克蛋白A (HspA)在食品级乳酸乳球菌NZ3900菌株中的克隆表达及免疫反应性.方法 利用基因重组技术构建乳酸乳球菌重组子NZ3900/pNZ8110-hspA;绘制生长曲线,观察hspA插入对乳酸乳球菌重组子生长影响及乳酸链球菌素(Nisin)对重组子生长影响；采用钠十二烷基硫酸盐-聚丙烯酰胺凝胶电泳法(SDS-PAGE)检测HspA在乳酸乳球菌中的表达；应用western-blot鉴定重组子表达HspA的免疫反应性.结果 成功扩增出幽门螺杆菌河南分离株(MEL-Hp27)的hspA基因,构建了hspA基因的乳酸乳球菌原核表达系统(NICE)；细菌生长曲线显示hspA的插入未对乳酸乳球菌重组子的生长产生影响,除10 ng/mL Nisin 对重组子生长影响较小外,20～100 ng/mL nisin对重组子的生长均有明显抑制；SDS-PAGE和Tricine SDS-PAGE 检测均未观察到HspA条带；western-blot鉴定结果显示乳酸乳球菌表达的HspA抗原蛋白具有良好免疫反应性.结论 HspA在乳酸乳球菌中的少量表达影响重组菌生长.     

点带石斑鱼过敏原分离、鉴定与纯化

目的 对点带石斑鱼的主要过敏原进行分离、鉴定与纯化.方法 通过十二烷基硫酸钠-聚丙炳酰胺凝胶电泳(SDS-PAGE)分离点带石斑鱼的蛋白质组份,采用免疫印迹(western-blotting)方法鉴定过敏原,通过离子交换层析对主要过敏原进行初步纯化.结果 SDS-PAGE结果显示,点带石斑鱼粗提液含有13条蛋白带,含量高的有8条,分子量分别为43,34,28,25,22,17,15,9 kD;westem-blotting显示,对点带石斑鱼过敏患者的阳性混合血清能与其中4个蛋白条带起反应,分子量分别为34,28,24,22 kD.离子交换层析可初步纯化出22 kD的过敏原蛋白.结论初步分离得到分子量为22 kD的点带石斑鱼主要过敏原,为临床诊断和治疗鱼类过敏性疾病提供理论依据.     

DEN-2分离株E基因部分序列原核蛋白表达

目的 通过构建登革2型病毒(DEN-2)临床分离株(B株)E基因区1-476bp的原核表达载体,进行原核表达,为DEN-2 E蛋白功能的研究提供基础依据.方法 将DEN-2 B株E基因区部分序列克隆入原核表达载体pET28a(+),命名为pET28a(+)-Eb;经酶切、PCR及测序鉴定后转化BL21(DE3)菌株,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,通过十二烷基硫酸钠-聚丙烯胺凝胶电泳(SDS-PAGE)、蛋白印迹(Western blot)鉴定表达蛋白;对表达蛋白进行纯化,并滴定对C6/36细胞的细胞半数致死量(TCID50).结果 成功构建了pET28a(+)-Eb原核表达重组质粒;SDS-PAGE分析表明,E基因区部分序列获得高效表达,其相对分子量约为23 kDa,表达量约占菌体总蛋白的29%;Western blot表明,该目的 蛋白可与DEN-2鼠单克隆抗体结合.用Ni柱亲和层析法纯化原核表达蛋白,纯度达90%.DEN-2 B株E基因部分序列原核表达蛋白对C6/36细胞的TCID50为10-5.31.结论 pET28a(+)-Eb可在BL21(DE3)菌株中高效表达;DEN-2 B株E基因部分序列原核表达蛋白对C6/36细胞有明显的细胞毒作用.     

中国龙虾变应原性组分分析

[目的]对中国龙虾(Panulirusstimpsoni)变应原性组分进行分离和鉴定。[方法]用Coca's液提取法制备龙虾变应原粗浸液,经聚丙烯酰胺凝胶电泳(SDS-PAGE)分离蛋白质组分,并用凝胶成像系统测定各组分的相对分子质量;采用蛋白质-印迹(Western-blotting)鉴定龙虾的主要及次要变应原。[结果]中国龙虾变应原粗浸液分离得到14种以上的蛋白质组分,有6种蛋白质能与患者血清特异性IgE结合,其中分子量为23000、34000、64000的蛋白质结合率较高。[结论]中国龙虾分子量为23000、34000和64000的蛋白质为主要变应原,分子量26000和64000两种蛋白质在粗浸液中含量并不高,但变应原性较强。     

辽宁省乙型脑炎病毒分离及进化树分析

目的 分离鉴定2007年辽宁省蚊虫携带流行型乙型脑炎(乙脑)病毒及基因型别.方法 用细胞培养方法分离病毒,对致金黄色地鼠肾细胞系(BHK-21)细胞病变(CPE)的病毒进行逆转录-聚合酶链式扩增(RTPCR)检测;扩增的目标片段进行序列测定;用Mega3.1软件、以邻位相连法构建进化树.结果 共分离到22株病毒,RT-PCR检测结果表明,均为乙型脑炎病毒;随机选择其中3株病毒的基因序列进行基因型鉴定分析,均为Ⅰ型乙型脑炎病毒.3株病毒E基因编码结构域(Domain)区段分析结果显示,其核苷酸和氨基酸同源性分别为99.3%和100%;与疫苗株SA14-14-2的Domain区相比,共有12个氨基酸位点变异.结论 辽宁省蚊虫标本中分离的病毒均为基因Ⅰ型乙型脑炎病毒,其中E基因所编码Domain区E-327(S→T)位点发生的变异,提示在辽宁省首次分离到乙型脑炎病毒变异株.     

口虾蛄原肌球蛋白基因表达及变应原性鉴定

目的 克隆口虾蛄原肌球蛋白(tropomyosin)基因并表达纯化出重组蛋白,研究其变应原性.方法 提取口虾蛄总RNA,设计特异引物,通过反转录聚合酶链反应克隆出目的 基因片段,测序后将该片段克隆到原核表达载体pET-28a上,转化到E coli BL21(DE3)后,经异丙基-B-D-硫代乳糖苷(IPTG)诱导表达,用Ni2+亲和层析柱对重组变应原进行纯化.采用免疫印迹(Western-blot)检测其与对虾过敏的患者血清IgE结合活性.结果 经序列测定,该基因含有长度为855bp的开放阅读框,编码284个氨基酸(GenBank登录号为EF584510).十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测该重组变应原在大肠埃希菌中高效表达36kDa的目的 蛋白,且重组变应原具有良好的IgE结合活性.结论 本研究成功获得了具有变应原活性的重组口虾蛄原肌球蛋白.     

2006年湖南省流行性感冒病原学监测结果与分析

目的了解湖南省流感监测地区的流感流行状况及流感毒株的型别分布,分析其流行趋势,为流感防制提供科学依据。方法采集流感样病例(ILI)的咽拭子标本,用传代狗肾细胞(MDCK)进行病毒分离,采用血凝抑制实验(HI)进行流感病毒型别鉴定。分离的毒株再送国家流感中心(NIC)进行复核鉴定。结果全省12家哨点医院3499份ILI咽拭子标本,分离到毒株258株,分离阳性率为7.37%,经NIC最后复核鉴定的结果为:A(H1N1)亚型202株,A(H3N2)亚型11株,B型43株,另有2株送NIC后转阴;流感及ILI暴发疫情病例咽拭子标本179份,分离到流感病毒75株,分离阳性率为41.90%,分型鉴定为A(H1N1)亚型12株,B型62株,1株送国家流感中心后转阴。结论2006年湖南省流感监测地区全年均有流感活动,A(H1N1)亚型、A(H3N2)亚型和B型均能被分离到,A(H1N1)亚型为优势株,而暴发疫情则以B型为主。     

大黄鱼过敏原的提取、分离及免疫学特性鉴定

目的提取、分离大黄鱼特异性过敏原成分,并对其免疫学特性进行鉴定。方法采用Coca’s提取液提取大黄鱼蛋白;十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析大黄鱼总蛋白的组成,以对鱼过敏患者阳性血清为一抗,Western-Blotting分析大黄鱼中的过敏原;阴离子交换层析对大黄鱼总蛋白进行初步分离,Western-Blotting分析各个组份的免疫学活性。结果大黄鱼粗浸液蛋白分子量在8～116kD之间;Western-Blotting检测到分子量为54、29、27和14kD的过敏原;通过离子交换层析分离,过敏原蛋白的相对含量有显著提高,且大多都保持原有的免疫学活性。结论研究发现了大黄鱼中多种过敏原并对其免疫学活性进行了鉴定。     

不同温度下鱼类食品过敏原免疫学特性分析

目的研究鱼类食品在生熟状态下的过敏原组分，分析其热稳定性过敏原。方法4种新鲜鱼类经不同温度处理后去鳞、内脏和鱼骨。在预冷丙酮中破碎，脱脂，Coca＇s液提取其生熟总蛋白。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离，考马斯亮蓝显色分析其总蛋白组成，用鱼过敏患者血清对不同温度条件下的鲩鱼总蛋白进行免疫学特征分析。结果SDS-PAGE显示，生熟鱼总蛋白提取物中均含有多种蛋白，经高温处理后熟鱼总蛋白组分相对减步。免疫印迹试验结果表明，加工温度、时间对鱼类食品过敏原有显著影响。结论鱼类食品中禽有热稳定的过敏原。即使加工后仍具有致敏性。     

Anaphylaxis induced by ingestion of raw garlic

Abstract Patients allergic to garlic often present dermatitis, rhinitis, asthma, and urticaria after ingestion of garlic, contact with garlic, or exposure to garlic dust. Garlic-related anaphylaxis is rare, and the impact of heating on garlic allergens is not very clear. We report a case of anaphylaxis induced by ingestion of raw rather than cooked garlic with manifestations different from previous reports, and we hypothesized that heating could reduce the allergenicity of garlic. Serum total immunoglobulin E (IgE) and specific IgE were tested using the Phadia CAP System FEIA (Phadia, Uppsala, Sweden). Protein extracts from raw and cooked garlic were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Serum-specific IgE for garlic was 8.16?kUA/L. IgE banding proteins could only be detected in raw garlic extract, because allergens in garlic were mostly degraded into small fragments after heating, as shown in SDS-PAGE profile. In conclusion, raw garlic could induce life-threatening anaphylaxis. However, most of its allergens are heat labile, and patients allergic to garlic might tolerate the cooked one well.     

Macrophage-stimulating activity of polysaccharides extracted from fruiting bodies of Coriolus versicolor (Turkey Tail Mushroom)

The macrophage-stimulating effect of polysaccharides extracted from Coriolus versicolor (Turkey Tail mushroom) was investigated, and their effectiveness was compared with that of lipopolysaccharide (LPS). The purified polysaccharide (CV-S2-Fr.I) of C. versicolor obtained by Sepharose CL-6B gel chromatography stimulated macrophage lysosomal enzyme activity by 250% at a concentration of 100 microg/mL, which was higher than that of LPS at the same concentration. When CV-S2-Fr.I was used in combination with interferon-gamma, there was a marked cooperative induction of nitric oxide production. However, CV-S2-Fr.I had no effect on nitric oxide production by itself. The proportion of C3-positive macrophages in the CV-S2-Fr.I group increased by 7.2-fold compared with the control group.     

Determination of unique microbial volatile organic compounds produced by five Aspergillus species commonly found in problem buildings

This study identified unique microbial volatile organic compounds (UMVOCs) produced by five Aspergillus species (A. fumigatus, A. versicolor, A. sydowi, A. flavus, and A. niger) cultivated on malt extract agar and gypsum board. The hypothesis was that UMVOCs can be used to predict the presence of Aspergillus species. During the cultivation humidified air was continually supplied and evenly distributed through each of the culture flasks. Volatile metabolites were collected using Tenax TA tubes on Days 8, 16, and 30 after inoculation. The volatile metabolites were determined by gas chromatography/mass spectroscopy after thermal desorption. Nine compounds recognized as UMVOCs--3-methyl-1-butanol; 2-methyl-1-propanol; terpineol; 2-heptanone; 1-octen-3-ol; dimethyl disulfide; 2-hexanone; 3-octanone; and 2-pentylfuran--were found on the cultures in detectable amounts. The first two compounds were detected at the highest frequency when combining both media. The first four compounds were found to be the dominant UMVOCs on gypsum board, which could be used as chemical markers of the common Aspergillus species grown indoors.     

染料亲和层析分离纯化酵母两种脱氢酶

目的探讨三嗪活性染料Cibacron Blue F3GA和KE-3B艳红制备的亲和层析柱用于建立分离纯化面包酵母乙醇脱氢酶和葡萄糖-6-磷酸脱氢酶的方法。方法在琼脂糖凝胶Sepharose 6B上,分别偶联三嗪活性染料Cibacron Blue F3GA和KE-3B艳红,制成染料亲和层析柱。将面包酵母经超声破碎、高速离心、硫酸铵分级沉淀、透析、冰冻干燥等步骤得到脱氢酶粗品,再经染料亲和层析柱进一步纯化。结果经过固定有Cibacron Blue F3GA的亲和层析柱,乙醇脱氢酶的纯化倍数为2.0,酶活性回收率为30.8%;经过固定有KE-3B艳红的亲和层析柱,葡萄糖-6-磷酸脱氢酶的纯化倍数为23.2,酶活性回收率为66.0%。所得的乙醇脱氢酶在SDS-聚丙烯酰胺凝胶电泳为1条带。结论染料配基亲和层析可作为面包酵母乙醇脱氢酶和葡萄糖-6-磷酸脱氢酶的精制纯化方法。     

Study on the quantitative and qualitative composition of fungi in dried medicinal plants

The quantitative and qualitative composition of fungi was determined in selected dried medicinal plants purchased in one of the herbal shops in Szczecin, Poland. The samples examined were as follows: chamomile (Flos Chamomillae), peppermint (Folium Menthae piperitae), lemon balm (Folium Melissae), St. John's wort (Herba Hyperici), and two herbal mixtures. The fungal composition depended on the specified sample. Xerophilic fungi, i.e. Eurotium amstelodami, E. herbariorum, E. rubrum and Wallemia sebi were isolated from dried medicinal plants. E. amstelodami was the predominating species. The prevailing thermophilic and thermotolerant species were Rhizopus microsporus var. rhizopodiformis and Aspergillus fumigatus. Pink and white yeasts were also numerous in some samples. Except for Aspergillus niger, mesophilic and toxigenous species were found to occur infrequently in the samples. However, Aspergillus versicolor was found to occur abundantly in lemon balm.     

Allergens from Fusarium solani identified by immunoblotting in asthma patients In Iran

We extracted Fusarium solani antigens to evaluate specific anti-F. solani IgE in fifty-one patients with asthma (33 men and 18 women) and in 22 non-atopic healthy subjects (15 men and 7 women). F. solani strains were cultured in Sabouraud glucose agar and subjected to cell disruption using the freeze-and-thaw method. The obtained cytoplasmic extracts were analysed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Sensitisation to F. solani antigens has been evaluated in asthmatic patients using the immunoblotting assay. The SDS-PAGE identified 29 protein bands in the cytoplasmic extracts of F. solani isolates, with molecular weights ranging from 24 kDa to 112 kDa. Immunoblotting detected specific anti-F. solani IgE antibody in all asthma patients, but not in the control group. The predominant reactive allergens in patients corresponded to the bands with molecular weights of 24 kDa, 58.5 kDa, 64.5 kDa, 69 kDa, 72 kDa, and 97 kDa. Our results suggest that various allergenic components of F. solani may produce symptoms of asthma in susceptible individuals and they call for further research.