转基因水稻表达的Bt毒蛋白CryⅠAb溶液构象的研究

用荧光光谱法研究了毒蛋白CryⅠAb在溶液中的构象. 结果表明, pH值增大时, 毒蛋白的荧光强度也增大, 至pH=11时荧光强度已达最大值;甲醇、乙醇及异丙醇等有机溶剂引起毒蛋白荧光发射波长蓝移和荧光强度增大, 至有机溶剂的浓度超过某一阈值后荧光发射波长不再蓝移;丙酮能完全猝灭毒蛋白CryⅠAb的荧光, 丙烯酰胺能猝灭CryⅠAb 86%的荧光, 而KI对毒蛋白的荧光无猝灭作用,由此推断CryⅠAb的大部分色氨酸残基位于分子表面富含负电荷的区域中.     

酶联免疫分析法探测Cry1Ab蛋白在不同介质中的构象变化

采用酶联免疫分析（ELISA）方法,根据构象变化后的蛋白与抗体结合能力下降从而导致ELISA测定值降低的原理,探测了转cry1Ab基因水稻表达的Cry1Ab蛋白在不同溶液介质中的热致构象变化行为,以及不同有机溶剂及溶液pH值对该蛋白构象变化的影响程度。实验表明,Cry1Ab蛋白在不同条件下的构象变化程度可以灵敏地通过ELISA方法检测。在不同的介质中,CrylAb蛋白的热致构象变化程度不同。在Na2SO4介质中,该蛋白具有较高的热稳定性;SDS的存在,可以促进该蛋白的构象变化。常温下,25％（V／V）的有机溶剂乙腈、异丙醇、甲醇、乙醇均能使该蛋白的构象发生转变,其中以乙腈最为显著。醇类溶剂对Cry1Ab蛋白的构象影响程度随疏水性增大而增大;溶液pH值也对该蛋白的构象变化产生影响。pH在8-10之间,该蛋白构象能保持稳定;酸或过碱性的溶液均能使蛋白构象偏离原始状态,从而引起ELISA测定值的降低。另外,腐殖酸能在一定时间内保持Cry1Ab蛋白构象的稳定性。     

转Bt基因植物表达产物Cry1Ab蛋白的制备纯化方法研究

以转Bt基因水稻为试材,研究其表达产物Cry1Ab蛋白的提取、分离及纯化的方法。实验结果表明,DEAE-纤维素填料对Bt蛋白有较好捕获效果。根据生物信息学方法预测了目标蛋白和主要共存蛋白的等电点和疏水性差异。合理地选择了阴离子交换色谱与疏水作用色谱组合方法。提取液经DEAE-Sephadex A-50柱层析及Phenyl-Sepharose Fast Flow疏水层析分离后,目标蛋白得到了显著的纯化。考察了疏水层析中用不同洗脱液洗脱Cry1Ab蛋白对活性回收率和纯度的影响,结果表明：以0．25mol／L KSCN作洗脱液对活性影响最小,HIC一步纯化倍数可达8倍,总纯化倍数达100倍。     

转基因水稻表达的Bt毒蛋白Cry I Ab溶液构象的研究

用荧光光谱法研究了毒蛋白Cry I Ab在溶液中的构象。结果表明，pH值增大时，毒蛋白的荧光强度也增大，至pH=11时荧光强度已达最大值；甲醇、乙醇及异丙醇等有机溶剂引起毒蛋白荧光发射波长蓝移和荧光强度增大，至有机溶剂的浓度超过某一阈值后荧光发射波长不再蓝移；丙酮能完全猝灭毒蛋白Cry I Ab的荧光，丙烯酰胺能猝灭Cry I Ab86%的荧光，而KI对毒蛋白的荧光无猝灭作用，由此推断Cry I Ab的大部分色氨酸残基位于分子表面富含负电荷的区域中。     

转基因作物pat蛋白的双抗体夹心酶联免疫检测方法研究

将克隆到的pat基因构建原核表达载体pET30(+)-pat并在大肠杆菌中进行表达,分离纯化得到高纯度pat蛋白。用所得到的pat蛋白制备了兔多克隆抗体和鼠单抗2F6。多抗效价>8000,单抗效价为5×104;单抗为IgG1类,轻链为κ型。基于抗pat蛋白单克隆抗体和兔多克隆抗体建立了双抗夹心酶联免疫分析方法(Sandwich ELISA)。检测范围为1.6~100μg/L,线性回归方程y=0.6914x-2.572,决定系数为0.9951。用所建立的方法测定了5个转基因抗虫棉和2个非转基因棉花品种叶片中pat蛋白的含量,其中转基因抗虫棉中的3个品种检测出pat蛋白,其余均未检出pat蛋白。     

W544F定点突变提高苏云金杆菌Cry1Ac蛋白的稳定性

W544是Cry1Ac蛋白上独特于其它Cry类蛋白的一个氨基酸, 它与F578和F604一起组成一个“螺旋桨状”的疏水簇, 通过疏水相互作用维持蛋白的三维结构稳定. 本研究通过定点突变将W544保守地替换为苯丙氨酸, SDS-PAGE分析结果表明其纯化的原毒素对紫外照射、胰蛋白酶处理和室温存贮的稳定性相对于野生Cry1Ac都有一定程度的提高; 经原子力显微镜观察, 发现W544F产生的晶体两个顶点间的垂直距离比野生型Cry1Ac约长0.6 μm, 且晶体表面不及野生型光滑; 此外, W544F与野生Cry1Ac的杀虫活性相似, 但经过紫外光照射9 h后, 其保留的杀虫活性比野生型高4倍以上. W544F突变较好地解决了Cry1Ac毒素蛋白田间应用不持久的问题, 具有重要的应用价值.     

超顺磁性免疫磁珠体系用于植物油中黄曲霉毒素B1的检测研究

利用化学共沉淀方法,制得了主要成分为Fe3O4的超顺磁性纳米磁珠。纳米磁珠和硅烷偶联剂氨丙基三乙氧基硅烷(APTES)反应而带上氨基基团。带上氨基的磁珠通过戊二醛活化后发生醛基化,与黄曲霉毒素B1(AFB1)多克隆抗体偶联得到黄曲霉毒素B1免疫磁珠。利用该免疫磁珠为净化工具,建立了有机溶剂萃取、免疫磁珠净化、高效液相色谱(HPLC)检测植物油中黄曲霉毒素B1的方法。该方法具有良好线性,线性范围为5～50μg/L,相关系数达0.999 4,检出限为0.5μg/L,平均回收率为96%,相对标准偏差为12.5%。该方法操作简单、灵敏度高、准确性好,可用于植物油中黄曲霉毒素B1的检测。     

基于磁珠的可见光检测微阵列信号的新方法

以磁珠作为标记物,提出了一种在微阵列上检测核酸的新方法。该法基于生物素同链霉亲和素的亲和作用,利用磁珠的超顺磁性和宏观可见特性,使得杂交结果可在普通的光学显微镜或放大镜下检测,甚至肉眼可见。以合成探针为对照,用参比荧光标记染料Cy3标记方法,对这种新方法的检出限进行了研究,并在此基础上采用大肠杆菌16s rDNA的PCR产物作为样品,进行了细菌检测的尝试,取得了较好的实验结果。本法所得的实验结果易于观测,无需采用大型的荧光检测仪器,因而大大降低了检测成本,与其它可见光检测方法(如金胶银染等)相比,具有方便、快捷的特点。这种新方法在传染病检测和环境监测中将具有广阔的应用前景。     

基于微磁珠分离技术的适配体实时定量PCR检测方法

利用适配体的识别能力和可扩增性, 构建了基于微磁珠分离技术的适配体实时定量聚合酶链式反应(PCR)检测方法. 通过微磁珠偶联的互补链与适配体序列之间的碱基配对结合, 有效除去溶液中未与靶分子结合的适配体序列, 采用实时定量PCR技术测定上清液中结合态的适配体序列浓度, 从而间接实现对靶分子的定量检测. 分别选取代表生物大分子和有机小分子的凝血酶和ATP作为检测对象, 验证了该方法的普适性. 研究结果表明, 在获取特异性适配体序列后, 仅需简单优化其互补链序列, 即可对超低含量的凝血酶和ATP进行准确定量, 检出限分别为50 pmol/L和5 μmol/L. 该方法具有同时适用于高特异性和高灵敏度地检测生物大分子和有机小分子的优势.     

Development and validation of a sensitive and fast chemiluminescent enzyme immunoassay for the detection of genetically modified maize

Proteins from the Cry 1 family, in particular Cry 1Ab, are commonly expressed in genetically modified Bt maize in order to control chewing insect pests. A sensitive chemiluminescent sandwich enzyme immunoassay for the detection of Cry1Ab protein from genetically modified Bt maize has been developed and validated. A Cry1Ab protein-specific antibody was immobilized on 96- or 384-well microtiter plates in order to capture the Cry1Ab toxin in the sample; the bound toxin was then detected by employing a second anti-Cry1Ab antibody and a horseradish peroxidase-labeled anti-antibody, followed by measurement of the enzyme activity with an enhanced chemiluminescent system. The chemiluminescent assay fulfilled all the requirements of accuracy and precision and exhibited limits of detection of a few pg mL⁻¹ Cry1Ab (3 or 5 pg mL⁻¹, depending on the assay format), which are significantly lower than that achievable using conventional colorimetric detection of peroxidase activity and also represent an improvement compared to previously developed Cry1Ab immunoassays. High-throughput analysis can be performed using the 384-well microtiter plate format immunoassay, which also allows one to reduce the consumption of samples and reagents. Validation of the assay, performed by analyzing certified reference materials, proved that the immunoassay is able to detect the presence of the Cry1Ab protein in certified reference samples containing as low as 0.1% of MON 810 genetically modified Bt maize. This value is below the threshold requiring mandatory labeling of foods containing genetically modified material according to the actual EU regulation.     

Binding properties of a monoclonal antibody against the Cry1Ab from <Emphasis Type="Italic">Bacillus Thuringensis</Emphasis> for the development of a capillary electrophoresis competitive immunoassay

Experimental work performed was aimed at the assessment of a competitive capillary electrophoresis immunoassay with laser-induced fluorescence (CEIA-LIF) detection for the determination of the Cry1Ab endotoxin from Bacillus thuringensis. The binding constant of a monoclonal antibody, raised against the insecticide protein Cry1Ab, was determined on a microplate by indirect enzyme-linked immunosorbent assay (ELISA) and compared with that obtained in-capillary under nonequilibrium separation conditions. The two binding constants appear comparable—(5.0 ± 1.2) × 10⁶ M⁻¹ and (9.06 ± 5.7) × 10⁶ M⁻¹—reflecting good preservation of the antibody binding behavior in the capillary electrophoresis format. These results allow use of a calibration curve possible between 0.2 and 150 nM of endotoxin protein, with a limit of detection of 0.5 nM (33 μg L⁻¹). Preliminary recovery experiments on maize extracts spiked with known amounts of Cry1Ab endotoxin also showed promising results in detecting the toxin in complex real matrices.     

Magnetic beads-based multicolor colorimetric immunoassay for ultrasensitive detection of aflatoxin B1

Aflatoxin B₁ (AFB₁) is one of the most toxic, mutagenic and carcinogenic mycotoxin, widely exists in contaminated food, grains and feedstuff products. In this study, a novel magnetic beads multicolor colorimetric immunoassay (MBMCIA) based on Au@Ag nanorods (Au@Ag NRs) is proposed to visual detect ultralow concentration of AFB₁ with high-resolution by the naked-eye. To design the MBMCIA system, AFB₁-BSA conjugates were first coated on the surface of magnetic beads (MBs), then alkaline phosphatase (ALP) as a bridge between immunoassay and color reaction was used for catalytic hydrolysis of ascorbic acid-phosphate to generate reductive ascorbic acid. Finally, the yielded ascorbic acid could reduce silver ions to grow a silver coating on the surface of gold nanorods to generate Au@Ag NRs, which leads to the bule-shifted longitudinal absorption peak of Au NRs, accompanying with a series of perceptible color change. Under the optimal conditions, the proposed MBMCIA exhibited good sensitivity and specificity for the detection of AFB₁ with the detection limit as low as 5.7 pg/mL. Meanwhile, the MBMCIA was also applied for the analysis of AFB₁ in spiked wheat samples, the obtained recoveries range from 99.1% to 104.3% with relative standard deviation (RSD) less than 7.05% were acceptable. The proposed MBMCIA integrates separated, enriched, anti-interference and signal read-out into one, which opens up a new avenue for an on-site visual food safety inspection or environmental monitoring.     

Graphene Oxide Conjugated Magnetic Beads for RNA Extraction

A magnetic material that consists of silica‐coated magnetic beads conjugated with graphene oxide (GO) was successfully prepared for facile ribonucleic acid (RNA) extraction. When the GO‐modified magnetic beads were applied to separate the RNA from the lysed cell, the cellular RNAs were readily adsorbed to and readily desorbed from the surface of the GO‐modified magnetic beads by urea. The amount of RNA extracted by the GO‐modified magnetic beads was ≈170 % as much as those of the control extracted by a conventional phenol‐based chaotropic solution. These results demonstrate that the facile method of RNA separation by using GO‐modified magnetic beads as an adsorbent is an efficient and simple way to purify intact cellular RNAs and/or microRNA from cell lysates.     

Development and validation of a sensitive enzyme immunoassay for surveillance of Cry1Ab toxin in bovine blood plasma of cows fed Bt-maize (MON810)

The increasing global adoption of genetically modified (GM) plant derivatives in animal feed has provoked a strong demand for an appropriate detection method to evaluate the existence of transgenic protein in animal tissues and animal by-products derived from GM plant fed animals. A highly specific and sensitive sandwich enzyme immunoassay for the surveillance of transgenic Cry1Ab protein from Bt-maize in the blood plasma of cows fed on Bt-maize was developed and validated according to the criteria of EU-Decision 2002/657/EC. The sandwich assay is based on immuno-affinity purified polyclonal antibody raised against Cry1Ab protein in rabbits. Native and biotinylated forms of this antibody served as capture antibody and detection antibody for the ELISA, respectively. Streptavidin-horseradish peroxidase conjugate and TMB substrate provided the means for enzymatic colour development.The immunoassay allowed Cry1Ab protein determination in bovine blood plasma in an analytical range of 0.4-100 ng mL⁻¹ with a decision limit (CCα) of 1.5 ng mL⁻¹ and detection capability (CCβ) of 2.3 ng mL⁻¹. Recoveries ranged from 89 to 106% (mean value of 98%) in spiked plasma.In total, 20 plasma samples from cows (n = 7) fed non-transgenic maize and 24 samples from cows (n = 8) fed transgenic maize (collected before and, after 1 and 2 months of feeding) were investigated for the presence of the Cry1Ab protein. There was no difference amongst both groups (all the samples were below 1.5 ng mL⁻¹; CCα). No plasma sample was positive for the presence of the Cry1Ab protein at CCα and CCβ of the assay.     

化学发光磁酶免疫法检测O157:H7大肠埃希菌

通过自行制备的免疫磁珠结合新型发光底物(AMPPD), 改进了化学发光磁酶免疫检测法, 对人工猪肉样品中O157:H7大肠埃希菌进行了检测, 并与人工计数法进行了比较.     

Magnetic beads-based immunoassay as a sensitive alternative for atrazine analysis

A new immunoassay strategy for sensitive atrazine determination based on magnetic beads is reported. The immuno-method is a competitive solid-phase immunoassay where the anti-atrazine antibody is immobilized on the magnetic beads surface and fixed at the reaction cell bottom using a simple magnet, which generates a magnetic field. Analyte and HRP (horseradish peroxidase) tracer compete for active sites of antibody. After the immunointeractions antibody-analyte and antibody-tracer, atrazine quantification from the sample is performed by injection of the chemiluminescence substrate (luminol, hydrogen peroxide and p-iodophenol). Different antibodies (polyIgG anti-atrazine Ab I and affinity purified polyIgG anti-atrazine AbI) were tested in this configuration. Also, optimum concentration of antibody-covered magnetic beads was set up (8 mg/l Ab II). Finally, the performance of magnetic beads-based immunoassay for atrazine determination was evaluated demonstrating that the magnetic beads-based immunoassay is one of the most sensitive method for atrazine determination (LoD = 3 pg/l, IC₅₀ = 37 pg/l, DR = 10-1000 pg/l).