苦荞过敏蛋白全长基因的克隆、表达及免疫学活性研究

荞麦属于一种常见的植物性过敏原。本实验在以往研究的基础上,以苦荞种子总RNA 为模板,通过RTPCR和5'-RACE 等方法,扩增、克隆获得苦荞过敏蛋白全长cDNA 序列。分析表明,该序列编码一个由515 个氨基酸残基组成的功能蛋白,并与甜荞过敏性贮藏蛋白的氨基酸序列同源性达到90% 以上。经原核表达及一步亲和纯化后得到纯度较高的重组苦荞过敏蛋白(rTBt)。采用竞争性ELISA 对其免疫活性分析显示,目的蛋白与荞麦过敏病人血清中的IgE 抗体可以特异结合。     

烟草C4H2基因的克隆与表达特性分析

以中烟202无菌幼苗为供试材料克隆出C4H2基因的CDS区,对该基因进行生物信息学分析,构建原核表达载体,在大肠杆菌中对重组质粒进行诱导表达及纯化分析,考查其在不同组织中的表达特性,以及叶片中黄酮含量变化趋势.结果表明:1)NtC4H2蛋白属于细胞色素P450超家族成员,有1个跨膜区,为不稳定亲水蛋白,二级结构中α螺旋...     

TRPV4原核表达纯化系统的构建及生物信息学分析

目的:构建非选择性阳离子通道蛋白TRPV4的原核表达纯化系统,并对其进行生物信息学分析,为与该目标蛋白相关疾病的研究和药物活性成分筛选提供技术支持.方法:将人源TRPV4基因分别克隆至pET-28a(+)、pET-32a、pET-15b、pGEX-5X-1、pEX-4T和pGEX-6p-1等6种表达载体,并利用BL21...     

植物乳杆菌WU14 6-磷酸-β-葡萄糖苷酶的基因克隆、蛋白表达及生物信息学分析

植物乳杆菌是一类兼性异养,兼性厌氧的同型发酵乳酸菌,其在改善食品风味和发酵特性等方面发挥着重要作用,而这种功能特征与其具有糖苷酶活性密切相关。为了挖掘植物乳杆菌糖苷酶的应用价值,以植物乳杆菌WU14为研究对象,利用聚合酶链式反应(PCR)技术成功克隆了8个WU14的糖苷水解酶,生物信息学分析表明均为糖苷水解酶1家族(GH1)的6-磷酸-β-葡萄糖苷酶基因。鉴于植物乳杆菌中该类糖苷酶鲜有报道,采用原核异源表达展开研究。序列分析表明8个酶蛋白氨基酸序列具有GH1家族的6-磷酸-β-葡萄糖苷酶典型的2个保守催化位点,序列一致性在32%～74%之间。经SDS-PAGE,8个蛋白均在大肠杆菌中表达,其中BglAW14、BglCW14、BglFW14为部分可溶性表达,其余为蛋白表达,形成包涵体。生物信息学分析显示:8个基因的编码蛋白都无信号肽和跨膜结构,疏水性较强。由亚细胞定位预测可知,除BglEW14主要存在于分泌结构内和BglHW14主要存在于细胞膜外,其余6个基因都存于细胞质内。对8个6-磷酸-β-葡萄糖苷酶基因的克隆表达及生物学序列分析,为进一步探究植物乳杆菌来源的6-磷酸-β-葡萄糖苷酶的分子机理研究奠定了基础。     

变形假单胞菌2-酮基葡萄糖酸激酶基因的克隆、表达及生物信息学分析

采用聚合酶链式反应技术,从2-酮基葡萄糖酸工业生产菌株变形假单胞菌JUIM01中克隆2-酮基葡萄糖酸激酶的全长基因kguK,并在此基础上构建了重组菌株大肠杆菌BL21(DE3)/pET-28a(+)-kguK。在异丙基-β-D-硫代半乳糖苷的诱导下,该重组菌株表达了一个特异的分子质量约为36.0 ku融合蛋白,且该蛋白的Western-Blot鉴定结果显示为阳性。生物信息学分析结果表明,该蛋白是一种由305个氨基酸残基组成的亲水性蛋白,定位于细胞质中,存在着与pfkB家族蛋白类似的保守结构域,其二级结构中α-螺旋、延伸链和无规则卷曲所占的比例分别为35.73%、12.79%和51.48%。     

啤酒酵母CRB2菌株乙醛脱氢酶基因(Ald6)的核苷酸序列分析、酶鉴定及在啤酒生产中的应用探讨

乙醛脱氢酶Ald6是啤酒酵母乙醛代谢途径的关键酶,它能催化乙醛脱氢,生成乙酸.设计引物,RT-PCR扩增得到啤酒酵母CRB2菌株Ald6基因,在大肠杆菌中进行克隆、表达、测序,并对表达产物的酶学性质进行了研究.结果表明,Ald6基因的大小为1503bp,编码的乙醛脱氢酶由500个氨基酸组成,与其它菌株的乙醛脱氢酶相比,核苷酸序列的同源性为7%～100%.乙醛脱氢酶以乙醛为底物时的Km值为28.14/μmol/L,Vmax为98.03μmol·min-1mg-1,最适反应条件为15℃,pH 8.0.中试酿造试验发现K 、Mg2 、Fe2 对降低乙醛含量有显著作用.     

Comparative study of in vitro digestibility of major allergen,tropomyosin and other proteins between Grass prawn (Penaeus monodon) and Pacific white shrimp (Litopenaeus vannamei)

BACKGROUND: Stability in simulated gastric fluid is supposed to be an important parameter for the estimation of food allergenicity. In the present study, the digestive stability of allergenic protein tropomyosin (TM) and other food proteins from Grass prawn and Pacific white shrimp in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) digestion assay system was investigated and comparatively studied by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE), western blotting, and inhibition enzyme‐linked immunosorbent assay (ELISA). RESULTS: In the SGF system, proteins such as actin and myosin heavy chain (MHC) were rapidly degraded within a short period of time, while TM was relatively resistant to pepsin digestion. In the SIF system, MHC was also easily decomposed, while TM and actin were resistant to digestion. Western blotting using a specific polyclonal antibody against TM indicated that the degradation pattern of shrimp TM by SGF and SIF was almost unaffected by the presence of other myofibrillar proteins. Further study by IgE immunoblotting and inhibition ELISA using sera from crustacean‐allergic patients indicated that IgE binding of TM was decreased. CONCLUSION: Proteinase digestion is effective in reducing IgE binding of shrimp TM. It is also of interest to notice that Pacific white shrimp TM had higher digestion stability than Grass prawn TM. However, Pacific white shrimp TM revealed enhanced IgE binding over that of TM from Grass prawn and thus it is possibly more allergenic. Copyright © 2010 Society of Chemical Industry     

Post mortem muscle protein degradation during ice‐storage of Arctic (Pandalus borealis) and tropical (Penaeus japonicus and Penaeus monodon) shrimps: a comparative electrophoretic and immunological study

Water‐, low‐salt‐ and high‐salt‐soluble protein fractions from the abdominal muscles of Pandalus borealis, Penaeus japonicus and Penaeus monodon extracted immediately after death and after 5, 16, 24, 48, 72, 96 and 120 h (P borealis) or 16, 22, 43, 71 and 92 h (Penaeus spp) of ice‐storage were analysed by one‐ and two‐dimensional electrophoresis and immunological techniques. The most evident effect in P borealis was the decrease in the relative amount of myosin heavy chain (MHC) and a concomitant increase in the number and intensity of bands of molecular size about 100 kDa cross‐reacting with anti‐MHC antiserum. MHC degradation of P borealis was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) of partially isolated native myosin. Other prominent features were the disappearance of bands of about 67 and 50 kDa after 24 h and the appearance of a band of slightly less than 50 kDa after 5 h of ice‐storage. These last bands showed the potential to be used as freshness markers. One spot tentatively identified as desmin did not suffer significant changes in any of the three species. Two bands (about 100 and 96 kDa) gave a positive reaction with the α‐actinin antibody in the zero‐time extract of P borealis, but after 24 h only one faint 96 kDa band was detected. In contrast, the extracts of P japonicus and P monodon did not suffer significant alterations during the examined period, and even after 92 h of ice‐storage only the 100 kDa anti‐α‐actinin cross‐reacting band was clearly visible in the high‐salt extract of P japonicus. © 2001 Society of Chemical Industry     

Purification, cloning, expression and immunological analysis of Scylla serrata arginine kinase, the crab allergen

BACKGROUND: Although crustaceans have been reported to be one of the most common causes of IgE‐mediated allergic reactions, there are no reports about the characterization and identification of arginine kinase (AK) from the mud crab (Scylla serrata) as allergen. In the present study, the purification, molecular cloning, expression and immunological analyses of the IgE allergen AK from the mud crab were investigated. RESULTS: The results showed that cloned DNA fragments of AK from the mud crab had open reading frames of 1021 bp, predicted to encode proteins with 356 amino acid residues. Sequence alignment revealed that mud crab AK shares high homology with other crustacean species. Mud crab AK gene was further recombined with the vector of pGEX‐4T‐3 and expressed in Escherichia coli BL 21. 2‐D electrophoresis suggested that native AK (nAK) and recombinant AK (rAK) shared the same molecular weight of 40 kDa, and the pI is 6.5 and 6.3, respectively. The nAK and rAK were further confirmed by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry. Immunoblotting analysis and colloidal gold immunochromatographic assay (GICA) using sera from subjects with crustacean allergy confirmed that the nAK and rAK reacted positively with these sera, indicating AK is a specific allergen of mud crab. CONCLUSION: Both of purified nAK and rAK reacted positively with sera from subjects with crustacean allergy in immunoblotting and GICA analysis, indicating AK is a common allergen of mud crab. In vitro expressed AK is proposed as a source of the protein for immunological or clinical studies. Copyright © 2011 Society of Chemical Industry     

建鲤组织蛋白酶L的克隆表达、纯化及活性特征鉴定

首先采用TA克隆技术克隆建鲤组织蛋白酶L（Cathepsin L,CAT L）成熟肽基因片段并进行双酶切鉴定,进而构建表达载体CAT L-p ET-30a并转入宿主菌E.coli BL21,经1 mmol/L异丙基-β-D-硫代吡喃半乳糖苷（IPTG）在37℃诱导2 h表达重组CAT L蛋白。而后经尿素梯度洗涤和镍离子亲和层析纯化目的蛋白,并利用SDS-PAGE检测诱导效果和纯化过程。最后以荧光合成肽底物（Z-Phe-Arg-MCA）测活法鉴定建鲤重组CAT L的热稳定性、p H稳定性,以及鱼糜生产和冻藏中常用添加剂对其活性稳定性的影响。双酶切鉴定结果表明成功克隆了目的基因片段,与鲤鱼CAT L基因序列相似性为99.11%。SDS-PAGE分析表明经诱导、尿素梯度洗涤及亲和层析后,成功获得高度纯化目的蛋白,分子量约28 ku。活性鉴定结果表明重组CAT L在20⁵0℃及p H3.0⁶.5范围内稳定;氯化钠、焦磷酸钠对重组CAT L活性的抑制作用呈现剂量依赖关系,而各浓度蔗糖、山梨醇则对其活性无明显作用。本研究成功克隆、表达和纯化了建鲤CAT L,并阐明了热、p H及鱼糜生产和冻藏中常用添加剂对该酶稳定性的不同影响。      

Purification,characterisation, and quantification of the soy allergen profilin (Gly m 3) in soy products

Profilins are pan-allergen proteins present in various plant foods and pollens. The objective was to develop a method for purification and characterisation of profilin from soy protein isolate. Furthermore, profilin was quantified in soy products and the effect of processing evaluated. Profilin was purified using poly-l-proline affinity chromatography, dialysis and ultrafiltration, and its quantification was implemented by indirect ELISA. Profilin in soymilks ranged from 4.37 ± 0.14 to 7.24 ± 0.30 mg/g protein, while in fermented products profilin ranged from 1.67 ± 0.02 to 5.47 ± 0.02 mg/g protein. Pasteurisation of soymilk was an ineffective method to completely eliminate profilin. Food matrix influenced thermal stability; at 100 °C, β-sheet and random coil structures were altered, while the α-helices remained intact. Induced fermentation of soybean meal by Bifidobacterium lactic, Lactobacillus plantarum and Saccharomyces cerevisiae resulted in 68.3% to 72.7% reduction of soy profilin. Heat treatment, fermentation and hydrolysis effectively reduced soy profilin.     

Impact of boron, calcium and genetic factors on vitamin C, carotenoids, phenolic acids, anthocyanins and antioxidant capacity of carrots (Daucus carota)

Carrots (Daucus carota L.) were used to investigate the effects and interactions of cultivar and mineral supply on the nutritional quality (antioxidant potential, vitamin C, carotenoids and phenolic acids) of the resulting storage roots. The supplement of boron (B) and or calcium (Ca) in the feeding solutions, during plant growth, influenced the accumulation of other minerals, such as P, K, Mg, S and Na, in the storage roots (p < 0.05). When no additional B or Ca was supplied (e.g. −B or −Ca treatment), we observed 33-50% increase in the accumulated levels of α- and β-carotenes, and 45-70% increase of vitamin C. Carrots grown with no supplement of B in the nutrient solutions (e.g. −B treatment and −ve control) had significantly higher (p < 0.001) levels of total phenolic acids compared to the carrots with the supplement of B (e.g. −Ca treatment and +ve control). A strong positive correlation was observed between the total phenolic contents and ORAC values (r = 0.932) in all the cultivars. The results suggest that both cultivar and mineral supply were major determinants of nutritional quality of the carrots. The nutritional value of carrot crops (with an acceptable physical quality) can be enhanced by manipulating mineral nutrient applications.