腰果主要过敏原Ana o 2原核表达及免疫学鉴定

目的 构建Ana o 2重组表达质粒,原核表达重组蛋白并评价其免疫活性。方法 将Ana o 2基因构建到pET-28a(+)载体中,测序正确的重组质粒转化Rosetta(DE3)感受态细胞,诱导表达目的蛋白并进行质谱鉴定。利用腰果过敏阳性血清,通过酶联免疫吸附试验(ELISA)和蛋白质免疫印记(Western blot)法评价重组Ana o 2的免疫活性。结果 重组蛋白进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)表明分子量为54 kD,与理论值相符,经质谱鉴定为Ana o 2。ELISA结果显示,用重组Ana o 2检测腰果过敏阳性血清与阴性血清特异性IgE抗体(sIgE)水平差异有统计学意义(t=2.44,P<0.05)。Western blot结果表明,重组Ana o 2与腰果过敏患者血清反应性良好。结论 利用原核系统表达了Ana o 2,并且重组Ana o 2与腰果过敏患者血清具有良好的反应性。     

鸡蛋主要过敏原Gal d 3基因的克降、表达、纯化及免疫学鉴定

目的:克隆表达鸡蛋主要过敏原Gal d 3基因并柃验其免疫活性.方法:提取鸡蛋的总RNA,采用RT-PCR方法扩增出目的基因片段,将其克隆入T载体中进行测序和分析.设计带有酶切位点的特异性引物,采用RT-PCR获得整个短的开放阅读框并将目的基因克隆到大肠杆菌表达载体pET-28a中进行诱导表达.通过Ni2+亲和层析柱对重组蛋白进行纯化,采用免疫印迹(Western blotting)方法检测其IgE结合活性.结果:克隆获得了鸡蛋主要过敏原Gal d 3.基因开放阅读框为2118个碱基(包括终止密码子),编码705个氨基酸.该序列编码的蛋白等电点为6.5,相对分子质量约为76000.表达产物经亲和层析纯化,用Western blotting方法检测免疫学活性,目的蛋白具有良好的免疫原性.结论:成功地克隆和表达了鸡蛋主要变应原Gal d 3基因,该蛋白具有良好的免疫原性.     

花生过敏原Ara h 6的分离纯化及鉴定

为高效分离纯化花生过敏原Ara h 6,通过脱脂、蛋白浸提、阴离子交换层析分离得到目的蛋白,并用十二烷基磺酸钠- 聚丙烯酰胺凝胶电泳(SDS-PAGE)、基质辅助激光解吸/ 电离飞行时间质谱(MALDI-TOF/MS)及免疫印迹技术(Western blotting)对其进行鉴定。结果表明,该蛋白为花生过敏原Ara h 6,其分子质量约为15kD,纯度大于95%,得率为22.5%。该方法简单、高效,可为花生过敏的进一步研究提供实验材料。     

牡蛎主要过敏原原肌球蛋白的重组表达及鉴定

牡蛎的主要过敏原是Crag 1蛋白(属于一种原肌球蛋白),克隆牡蛎原肌球蛋白Crag 1基因,导入大肠杆菌(BL21)中,然后筛选重组菌株。通过SDS-PAGE探究了重组菌株中Crag 1蛋白的最佳诱导表达条件,15℃下诱导16h。然后通过Western印迹、SDS-PAGE、质谱(MALDI-TOF-MS)、圆二色(CD)光谱等方法从His-tag、相对分子量、肽段比对以及二级结构上综合鉴定了重组Crag 1蛋白的准确性。本实验成功重组表达并鉴定了牡蛎主要过敏原Crag 1蛋白,为接下来的牡蛎脱敏实验以及牡蛎过敏的临床诊断和治疗奠定了良好的生物学基础。     

过敏症豚鼠模型的建立及海虾主要过敏原组分的纯化与鉴定

旨在建立海虾过敏症豚鼠模型,分析主要过敏原蛋白质组分并将其应用ELISA来提高体外检测的准确性。以海虾蛋白浸液为致敏原,氢氧化铝为佐剂免疫豚鼠,建立豚鼠过敏模型,DEAE阴离子交换纯化主要的过敏原成分,间接ELISA法检测sIgE和IgG。实验结果:模型组血清sIgE和IgG效价分别为18∶0和12∶0480;DEAE阴离子交换层析成功纯化出了海虾中的主要过敏原成分,应用于ELISA检测sIgE效价为13∶20;主要过敏原蛋白检测sIgE效价是蛋白浸液用于检测的4倍。结果表明,海虾过敏症豚鼠模型建立成功,并且纯化的海虾过敏原组分应用于ELISA检测能提高sIgE的检测水平,对海虾过敏症的体外诊断和治疗有一定的意义。     

腰果主要过敏原Ana o 3的重组表达与鉴定

目的克隆获得腰果主要过敏原Ana o 3基因,并利用pCold-SUMO原核表达载体重组表达Ana o 3并鉴定免疫活性。方法提取腰果总RNA,逆转录至c DNA,设计特异性引物,通过巢式PCR技术克隆腰果Ana o 3基因,将其插入pCold-SUMO vector,鉴定并测序;将测序正确的阳性质粒转化大肠埃希菌BL21(DE3),低温15℃诱导表达。摸索诱导表达条件,经镍柱纯化,并通过Western blot鉴定免疫活性。结果测序结果表明,克隆腰果Ana o 3基因片段全长为417 bp,与GenBank上Ana o 3基因CDS序列基本一致;十二烷基硫酸钠聚丙烯酰胺凝胶电泳结果表明,目的蛋白分子量为27 kD左右,大小与理论值相符;Western blot结果表明,与腰果过敏阳性血清具有良好的反应性。结论从腰果中成功克隆了Ana o 3基因,并于原核系统表达了Ana o 3蛋白,证实了此蛋白与腰果过敏血清具有良好的反应性。     

小龙虾主要过敏原精氨酸激酶的表达、纯化及 免疫原性鉴定

目的对纯化后的原核表达小龙虾主要过敏原蛋白精氨酸激酶进行免疫学鉴定。方法将化学合成的精氨酸激酶基因克隆至pET-28a(+)表达质粒上,转化进入大肠杆菌Rosetta(DE3)中,使用异丙基硫代半乳糖苷进行目的蛋白的诱导表达,用Ni~(2+)亲和层析柱对重组过敏原蛋白进行纯化,使用小龙虾过敏病人混合血清对纯化后的蛋白进行免疫印迹鉴定。结果纯化得到了分子量大小约为40 kDa的重组小龙虾精氨酸激酶,并且重组蛋白与过敏病人血清IgE有明显的特异性结合。结论本实验建立了小龙虾精氨酸激酶的原核表达方法,并鉴定了其免疫原性,为小龙虾过敏的基础研究、临床诊断与治疗奠定了基础。     

豇豆血红蛋白Lb II在大肠杆菌中的重组表达条件优化、纯化与鉴定

豇豆根瘤中含有大量豆血红蛋白。该蛋白是良好的天然色素,可用于人造肉的着色,本文将携带豇豆血红蛋白Lb II基因的质粒pET-15b转化进入大肠杆菌BL21中表达,并对表达条件进行优化。通过查找NCBI数据库得到一条全长456 bp的豇豆血红蛋白Lb II的序列,以pET-15b作为表达载体,大肠杆菌BL21-CodonPlus （DE3）-R-IL作为重组工程菌进行表达,成功表达出豇豆血红蛋白Lb II,并使用镍柱层析对蛋白进行初步纯化,同时添加抗坏血酸为抗氧化剂。以IPTG浓度、温度、时间为自变量,Lb II表达量为因变量进行单因素实验。结果表明,在IPTG终浓度为1.0 mmol/L、诱导温度为25 ℃,时间为14 h时,重组豆血红蛋白Lb II的表达量最高,经SDS-PAGE凝胶电泳和可见光光谱分析法鉴定为目的蛋白Lb II。经响应面试验优化后,表达量可以达到7.30 μg/mL。本研究为后续使用工程菌发酵生产豆血红蛋白奠定了基础。     

重组类人Ⅰ型胶原蛋白肽在大肠杆菌中的表达纯化及功能鉴定

本研究构建了编码重组类人Ⅰ型胶原蛋白肽基因的原核表达载体pET28a-rhCⅠ,并将其转化到大肠杆菌Rosetta(DE3)中进行诱导表达。采用PCR的方法扩增重组类人Ⅰ型胶原蛋白肽的cDNA序列,并将其克隆到原核表达载体pET28a上;重组质粒转化到大肠杆菌Rosetta(DE3)中进行IPTG诱导表达,并优化表达条件。利用SDS-PAGE和WesternBlot检测表达产物。大量表达重组蛋白,采用镍亲和层析进行纯化,并对纯化后的蛋白进行体外抗氧化研究以及促细胞增殖分析。结果表明,通过大肠杆菌Rosetta(DE3)诱导表达获得了分子量约为40ku的重组蛋白,与预期相符。经镍亲和层析获得纯度较高的蛋白,通过DPPH实验证明其有一定的抗氧化活性;而且MTT实验证实该蛋白可以促进小鼠成纤维细胞3T3细胞的增殖,为其在食品、化妆品和医疗行业的应用提供一定的理论依据。     

苦荞10 kD过敏原的重组表达及部分性质分析

以苦荞种子灌浆期cDNA文库中获得的苦荞10 kD过敏原基因序列TBAP10（tartary buckwheat 10 kD allergenprotein,TBAP10;GenBank登录号JK729379.1）为基础,构建重组表达载体pET47b-TBAP10,重组蛋白在大肠杆菌BL21 Star（DE3）中以包涵体形式表达。经包涵体复性和钴离子螯合层析纯化目的蛋白,并对其过敏活性、热稳定性及在模拟胃肠环境中的稳定性进行了分析。Western blotting显示该蛋白与苦荞16 kD过敏蛋白Fag t2存在免疫交叉反应。竞争性ELISA证明重组蛋白TBAP10具有与荞麦过敏患者血清IgE特异的结合活性;TBAP10具有强的热稳定性,能耐受15 min的沸水浴;模拟胃肠环境的消化结果显示TBAP10对胃蛋白酶具有强的耐受性,但对胰蛋白酶无耐受性。     

Immunoglobulins from Egg Yolk: Isolation and Purification

Simple water dilution was employed for the separation of water-soluble plasma proteins from egg yolk granules. An optimum recovery of immunoglobulin Y (IgY, 93–96%) in water-soluble fraction was obtained by sixfold water dilution at pH between 5.0–5.2 with incubation time of 6 hr at 4°C. Among the factors studied, pH was found to be the most important factor affecting IgY recovery. Active IgY of high purity with good recovery was obtained by a combination of several techniques including salt precipitation, alcohol precipitation, ultrafiltration, gel filtration and ion exchange chromatography. Salt precipitation, ultrafiltration, and get filtration is the recommended sequence. Over 100 mg of electrophoretically pure IgY was routinely obtained from one egg.     

Purification of Antibodies from Industrially Separated Egg Yolk

Industrially separated egg yolk was diluted and water soluble proteins separated by sedimentation. The supematant was filtered and applied to a column packed with cation exchanger within an automated liquid chromatography system. This was scaled-up from a 50 mL to a 1500 mL column. Two cation exchangers were investigated and immunoglobulin recoveries of 60–65% were obtained with 60–69% purities. Batch separation resulted in lower recovery (55%) and purity (57%). Further purification was investigated using anion exchange chromatography and salt precipitation. Results were improved with one step salt precipitation where purity was increased. The process is simple, economical and should prove useful for large production of IgY.     

Volatiles and Sensory Characteristics of Cooked Egg Yolk, White and Their Combinations

Headspace volatiles were produced by heating fresh egg yolk, white and different ratios of yolk to white. Volatiles of the same treatments produced during steam distillation extraction were identified, compared, and related to sensory characteristics of fresh scrambled eggs. Overall impression, sulfur, and sweet notes were sensory characteristics identified to distinguish between samples with varying yolkwhite ratios. Volatile concentrations of compounds in the headspace, and steam distillation/ solvent extracts were used to distinguish between scrambled eggs with different yolk:white ratios. The concentration of both egg yolk and white had a significant effect on fresh scrambled egg sensory characteristics and flavor volatiles. The contribution of both must be considered when producing egg substitutes.     

蛋白质量浓度对鸡蛋热诱导凝胶特性的影响

以蛋清、蛋黄和全蛋液为原料,研究蛋清、蛋黄和全蛋蛋白热诱导凝胶的形成能力以及蛋白质量浓度对蛋清、蛋黄和全蛋蛋白热诱导凝胶特性的影响。结果表明：蛋清、蛋黄和全蛋形成热诱导凝胶的最低蛋白质量浓度分别为50、55、50mg/mL;在50～135mg/mL范围内,随蛋白质量浓度的增加,蛋清、蛋黄和全蛋蛋白的凝胶强度和保水性不断增大,蒸煮损失整体呈下降趋势;蛋白质量浓度对蛋清、蛋黄和全蛋凝胶的弹性也有显著影响。     

Heat Denaturation and Emulsifying Properties of Egg Yolk Phosvitin

Phosvitin in water at pH 7 had a denaturation temperature (T_d) of 79.7 ± 1.4°C when heated at 10°C/min. When dissolved in 0.1M and 1.0M NaCl, the T_d decreased to 77.7 ± 1.2°C and 77.2 ± 1.3°C, resoectivelv. and in 10 and 20% sucrose there was no change in T_d. Heat treatment of phosvitin solutions at ≥65°C led to decreased emulsifying activity (EA). The emulsion stability (ES) decreased when phosvitim solutioni were heated at 70, 80 or 96°C for up to 60 min. The ES was not affected (p < 0.05) for phosvitin solutions after heating at ≤67.5°C for up to 60 min.     

Composition, Solubility and Emulsifying Properties of Granules and Plasma of Egg Yolk

Yolk was fractionated by a low speed centrifugation into granules and plasma. The composition, solubility and emulsifying properties of granules and plasma were compared to those of industrial spray-dried yolk. Granules contained about half the lipids and cholesterol and about double the proteins of yolk and plasma. Yolk and granules required an ionic strength ≥ 0.3M sodium chloride to become solubilized at pH 7.0, whereas plasma was solubilized at any ionic strength. At about 80% solubility, yolk, granules and plasma had similar emulsifying activities and granules had the best emulsion stabilization. Results suggest that granules could be used as stabilizers in food emulsions.     

从蛋黄中提取及纯化卵磷脂

本文以鸡蛋的卵黄为原料,采用含水乙醇与乙醚提取,通过丙酮脱水去油,得到粗品卵磷脂;进一步以氯仿/甲醇为洗脱液,硅胶柱层析法分离纯化自制的粗品卵磷脂,得到高纯度的纯白色固体卵磷脂,由薄层色谱定性检测,仅含一个斑点;用薄层扫描仪定量检测纯度大于97.4%。     

花生过敏原蛋白Ara h 6基因克隆和原核表达

本实验首先从花生中提取总RNA,利用反转录聚合酶链式反应技术克隆了花生过敏原蛋白Ara h 6全cDNA,并以此为模板扩增出Ara h 6目的基因。将目的基因与pMD19-T Simple质粒进行重组后转入BL21（DE3）宿主表达菌中,异丙基-β-D-硫代吡喃半乳糖苷诱导产物表达,并利用镍离子亲和层析纯化表达产物。DNA测序结果显示Ara h 6基因片段全长为438 bp,编码145 个氨基酸,与已知该蛋白DNA序列97%相同;十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果显示表达产物分子质量为24 kD,与融合组氨酸标签的重组Ara h 6蛋白理论分子质量相符;质谱鉴定结果表明重组蛋白的一级结构与天然Ara h 6匹配度为100%;Western blotting结果显示融合蛋白能够为抗Ara h 6多克隆抗体所识别,具有免疫原性。