杂色曲霉菌变应原分离、鉴定及纯化

目的 对杂色曲霉菌变应原蛋白进行分离、鉴定与纯化.方法 提取杂色曲霉菌蛋白质,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离,用免疫印迹(western-blot)鉴定变应原成分,用western-blot法鉴定出了6种变应原,通过离子交换和凝胶过滤层析纯化变应原.结果 碳酸氢盐-盐水提取液(Coca'...     

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。     

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．     

腰果主要过敏原分离鉴定与纯化

目的 对腰果的主要过敏原进行分析、鉴定与纯化。方法 通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)电泳分离腰果的蛋白质组份,采用免疫印迹(Western-blotting)方法鉴定过敏原,通过离子交换层析对腰果主要过敏原进行初步纯化。结果 腰果粗提液SDS-PAGE显示有10条蛋白条带,Western-blotting显示对腰果过敏患者的阳性混合血清能与其中2个蛋白条带起反应,分子量分别为21kD和11kD。离子交换层析可初步纯化出21和11kD的过敏原蛋白。结论 对腰果过敏原成功进行分离和鉴定,并初步纯化出腰果的主要过敏原。     

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43,34,28,25,22,17,15,9kD;Western-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34,28,24,22kD.离子交换层析可初步纯化出22kD的过敏原蛋白.结论 初步分离得到分子量为22kD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.     

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。     

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。     

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.coliBL21(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。     

食蟹猴精原干细胞分离纯化及鉴定

目的掌握食蟹猴精原干细胞 (spermatogonial stem cells,SSCs) 体外培养生长特性,建立食蟹猴精原干细胞分离、纯化、培养及初步鉴定的方法。 方法经手术获取2岁14天食蟹雄猴单侧睾丸,采用三步酶消化法分离获得单细胞悬液和差异贴壁法富集精原干细胞。将细胞培养于经丝裂酶素C处理的STO细胞层上,使用添加神经胶质细胞源性的神经营养因子(GDNF)、碱性成纤维细胞生长因子(bFGF)、GDNF家族受体α (GFRa1) 三种重要生长因子的无血清培养液体外培养,20d后采用CDH1标记分子免疫荧光染色和RT-PCR对培养的食蟹猴细胞进行SSCs初步鉴定。 结果通过差异贴壁法分离纯化富集的SSCs,接种到丝裂霉素C处理的STO饲养层细胞上,第2天开始分裂增殖。用含有生长因子的培养基培养2 d细胞形成小集落,5 d后细胞集落明显。培养20 d后,SSCs呈葡萄串状细胞簇,符合SSCs的形态特征,这些细胞经CDH1标记分子免疫荧光染色和RT-PCR均呈阳性表达。 结论本研究成功建立食蟹猴精原干细胞的分离纯化及鉴定体系。基于STO饲养细胞的添加GDNF、bFGF和GFRa1三种生长因子的无血清培养体系可用于食蟹猴精原干细胞培养,CDH1可作为食蟹猴精原干细胞鉴定的标志物。     

Purification design and practice for pertactin,the third component of acellular pertussis vaccine,from Bordetella pertussis

Development of acellular pertussis vaccine (aPV) requires purification of several components from Bordetella pertussis. While the components pertussis toxin (PT) and filamentous hemagglutinin (FHA) have been successfully purified, the third component, pertactin, proves to be a difficult target due to its very low concentration. In order to solve its purification problem, we performed the surface potential analysis with GRASP2 program. The results demonstrated that there are two major charge patches, one negative and one positive, which are located separately on this linear protein. For this special feature, we designed a dual ion exchange chromatography strategy including an anionic exchange and a cationic exchange process for separation of pertactin from the heat extract of B. pertussis. The initial anionic exchange chromatography concentrated the product from 1.7% to 14.6%, with recovery of 80%. The second cationic exchange chromatography increased the purity to 33%, with recovery of 83%. The final purification was accomplished by hydrophobic interaction chromatography, yielding a purity of 96%. The total recovery of the three columns was 61%. Characterization of the purified antigen was performed with CD, intrinsic fluorescence, HP-SEC and western-blot, showing that the purified protein kept its natural conformation and immune-reactivity. The rationally designed process proved to be feasible, and it is suitable for large-scale preparation of the third aPV component pertactin.     

Characterization of size, structure and purity of serogroup X Neisseria meningitidis polysaccharide, and development of an assay for quantification of human antibodies

Serogroup X Neisseria meningitidis (MenX) has recently emerged as a cause of localized disease outbreaks in sub-Saharan Africa. In order to prepare for vaccine development, MenX polysaccharide (MenX PS) was purified by standard methods and analyzed for identity and structure by NMR spectroscopy. This study presents the first full assignment of the structure of the MenX PS using (13)C, (1)H and (31)P NMR spectroscopy and total correlation spectroscopy (TOCSY) and (1)H-(13)C heteronuclear single quantum coherence (HSQC). Molecular size distribution analysis using HPLC-SEC with multi-angle laser light scattering (MALLS) found the single peak of MenX PS to have a weight-average molar mass of 247,000g/mol, slightly higher than a reference preparation of purified serogroup C meningococcal polysaccharide. MenX PS tended to be more thermostable than serogroup A PS. A method for the quantification of MenX PS was developed by use of high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). A novel and specific ELISA assay for quantification of human anti-MenX PS IgG based on covalent linkage of the MenX PS to functionally modified microtitre plates was developed and found valid for the assessment of the specific antibody concentrations produced in response to MenX vaccination or natural infection. The current work thus provides the necessary background for the development of a MenX PS-based vaccine to prevent meningococcal infection caused by bacteria bearing this capsule.