量子点探针应用于粮油中黄曲霉毒素免疫检测的研究

为改良检测技术,拓宽量子点探针在农产品安全领域的应用范围,制备了用于粮油中黄曲霉毒素检测的2种量子点探针,即碳量子点(CQD)探针和石墨烯量子点(GQD)荧光免疫探针,研究2种探针应用于黄曲霉毒素检测中的可行性。采用绿色合成方法,发现以0.375g/m L柠檬酸水溶液中获得的石墨烯量子点荧光强度最强。利用1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC·HCL)和N-羟基琥珀酰亚胺(NHS),将碳量子点和GQD与抗黄曲霉毒素的单克隆抗体1C11进行共价偶联,发现偶联前后量子点的荧光光谱变化趋势一致,而且偶联后均保持了较好的荧光特性,因此均可作为黄曲霉毒素免疫检测的探针,且与抗体偶联后的碳量子点的荧光特性优于石墨烯量子点。     

黄曲霉毒素抗原分子中半抗原偶联比对免疫分析灵敏度的影响

为提高黄曲霉毒素的检测灵敏度,探明黄曲霉毒素抗原分子中半抗原偶联比对免疫分析灵敏度的影响。采用黄曲霉毒素M1与羧甲氧基羟氨半盐酸盐进行肟化反应生成半抗原,再经活泼脂法,将半抗原与牛血清白蛋白（BSA）脱水反应实现偶联,生成自制人工抗原AFM1-BSA。采用竞争ELISA法、荧光与紫外光谱法鉴定,结果均证明自制人工抗原AFM1-BSA合成成功。进一步通过摩尔消光系数测算得AFM1的自制和商业抗原偶联比分别为0.7和6.3,自制抗原偶联比仅是商业抗原的1/9,二者偶联方法相同,但偶联比差异显著,为进一步研究比较提供了可能。将自制人工抗原AFM1-BSA和商业抗原AFM1-BSA分别与实验室自制的系列黄曲霉毒素单抗1C11、2C9、LM13、LM47反应,结果显示四种抗体对商业抗原均有很高的灵敏度,只有2C9对自制人工抗原有较高的灵敏度（1.997 ng/mL）,明显低于商业抗原作为包被抗原与2C9反应时的灵敏度（0.047 ng/mL）,说明针对同一种毒素AFM1制备的抗体,高偶联比抗原有助于提高免疫分析灵敏度。研究结果为AFM1高灵敏免疫检测技术研究提供了科学依据。     

高特异性黄曲霉毒素B1单克隆抗体的 制备及特性研究

本研究将黄曲霉毒素B1转化为其半缩醛B2a,在硼氢化钠（NaBH4）还原作用下与载体蛋白偶联制备完全抗原。将制备的完全抗原免疫Balb/c小鼠,经4次免疫后取其脾脏与小鼠骨髓瘤细胞Sp2/0细胞融合,采用半固体培养基筛选后鉴定,获得杂交瘤细胞株3A12,抗体的灵敏度可达6.1±0.025ng/mL,抗体与其它黄曲霉毒素B2、G1及G2的交叉反应率依次为7.8%、20.2%及0.6%,与黄曲霉毒素M1交叉反应率小于0.1%。为研发花生等农产品黄曲霉毒素B1特异性免疫分析技术及产品奠定了重要基础。     

量子点标记荧光免疫法检测花生中黄曲霉毒素B1

建立了基于量子点标记二抗的间接竞争荧光免疫吸附测定方法（indirect competitive fluorescence-linked immunosorbent assay,cFLISA）,并研究检测花生中黄曲霉毒素B1可行性。采用谷胱甘肽为稳定剂,在水相中直接合成碲化镉（CdTe）量子点,并利用EDC与兔抗鼠二抗进行共价偶联,以黄曲霉毒素B1的单克隆抗体建立cFLISA方法。结果表明,该方法的灵敏度和最低检测限值分别为0.023ng/mL和0.001ng/mL,与传统的有机染料FITC-二抗法比较,灵敏度提高了30倍,花生样品加标0.1、0.05和0.025ng/g,回收率范围在88%~116%之间,变异系数均小于10%。建立的cFLISA方法可以较好的检测花生中黄曲霉毒素B1,并为其它真菌毒素的检测提供参考。     

黄曲霉毒素的发生危害与防控方法

黄曲霉毒素是黄曲霉真菌的代谢化合产物,发生范围分布世界各地,通过感染食物和饲料,致使家禽、家畜及人类发生黄曲霉中毒及各种并发疾病,是目前人们认识最多、研究最广的一种微菌毒素.黄曲霉毒素的发生受外界环境因素的作用,污染程度主要受地理环境、农业生产栽培及收后储藏与加工过程等环节影响.由于黄曲霉毒素对人类和动物具有潜在致癌性,因而受到特别的关注,许多国家对用作食物和饲料的农产品给出黄曲霉毒素最低含量限定指标,以此减轻黄曲霉毒素对人类健康的危害.     

大豆蛋白偶联壳聚糖微球介质提取纳豆激酶的研究

采用液体反相悬浮法制取壳聚糖微球,在微球表面偶联纳豆激酶的亲和配体一大豆蛋白,作为纳豆溶栓酶的亲和吸附介质.结果表明:壳聚糖微球本身对纳豆激酶没有特异吸附,偶联大豆蛋白的壳聚糖微球对发酵粗酶液中的纳豆激酶的吸附性能符合Langmuir方程,吸附平衡时间约为60 min,最大吸附量为3 926.56 U·8-1.纳豆激酶...     

抗黄曲霉毒素单克隆抗体F(ab')2片段的制备与活性

为剧毒靶标(黄曲霉毒素)绿色分析提供高效抗体,在已有抗黄曲霉毒素杂交瘤细胞株1Cll的基础上,通过小鼠腹水法制备单克隆抗体,经胃蛋白酶酶解,制备F(ab')2片段,发现在优化条件37C、pH4.1柠檬酸缓冲液中酶解4.5h,可高效制备F(ab')2片段.通过酶联免疫吸附法(ELISA)比较了抗体片段与原始抗体的识别活性,发现F(ab')2片段效价达到1∶320000,是原抗体效价的1.5倍;灵敏度(IC50)为8.7pg/mL,保持了原抗体的抗原结合能力.     

抗黄曲霉毒素单链抗体在毕赤酵母X-33中的表达

为降低黄曲霉毒素单链抗体制备成本,提高单链抗体表达活性,利用获得的含抗黄曲霉毒素scFv基因的重组质粒pCANTAB 5E-scFv1A7,克隆出了scFv基因片段,并构建了酵母表达载体pPICZαA-scFv1A7,利用SacⅠ酶将重组载体线性化后插入到毕赤酵母X-33染色体基因组中,构建了pPICZαA-scFv1A7 X-33重组酵母,用0.8%甲醇诱导其分泌表达成功。间接竞争ELISA方法检测到该scFv对黄曲霉毒素B1的抑制率(IC50)值为4.5ng/m L,表明重组酵母表达产物scFv具有很好的抗原结合活性,可用于黄曲霉毒素的检测。     

抗黄曲霉毒素B1单链抗体(scFv)库的建立和筛选

从抗黄曲霉毒素B1单克隆抗体细胞系中扩增到重链可变区(VH)和轻链可变区(VL),经PCR将VH、Linker和VL连接成单链抗体(scFv),并克隆到载体pCANTAB-5E,经电转化大肠杆菌TG1,构建了库容量为1×108的单链抗体库.通过辅助噬菌体M13K07感染后,使单链抗体展示在噬菌体衣壳蛋白pⅢ的N端,得到客容量为1×108的噬菌体展示的抗体库.将抗体库用于"淘洗-富集-扩增"4轮以后,得到针对AFB1特异性的噬菌体抗体.进一步以噬菌体感染大肠杆菌HB2151,在细菌细胞周质中可溶性大量表达,最终经竞争EUSA分析表明获得了3株可以稳定分泌抗黄曲霉毒素B1 scFv菌株,分别命名为3C3、383和4A2.     

抗黄曲霉毒素单链抗体基因的克隆与表达

为降低黄曲霉毒素大量样品的制备成本,在实验室已有的抗黄曲霉毒素单克隆抗体8F6的基础上,成功克隆得到了该单克隆抗体的重链（VH）和轻链可变区（VL）基因片段。通过重叠延伸PCR的方法将轻、重链可变区基因连接,并引入连接肽(Linker) 编码序列,构建VH-Linker-VL结构的单链抗体(ScFv)基因, 并将该基因克隆到噬菌体表达载体pCANTAB 5E 上,使单链抗体以噬菌体展示形式在大肠杆菌TG1 中表达。间接竞争ELISA方法检测到该ScFv对黄曲霉毒素B1的抑制率（IC50）值为0.57ng/mL,表明该单链抗体与亲本鼠单抗有相同的抗原结合特异性,且具有很高灵敏度。     

茉莉酸在橡胶树体内移动的放射自显影研究

以巴西橡胶树（HeveabasilkensisMuLAg）无性系ZY7-3-97萌条为材料,采用石蜡切片的放射自显影技术,研究外源茉莉酸在植物体内的移动规律。对阳-A喂饲的材料采用2%（质量浓度）EDC（1-乙基-3-B3-（二甲胺基）丙基】碳二亚胺盐酸盐）+4%（体积分数）戊二醛（0.1molL.磷酸盐缓冲液,pH7.2）固定24h,进行石蜡切片的放射自显影。结果表明,在1mh内,施用部位的表皮、皮层、韧皮部和木质部中均有粗-JA 分布;5~30mn内,在施用部位及其上下5cm和10cm 处的茎中,木质部和韧皮部中均含有粗-JA,但在表皮和皮层薄壁细胞中则无。阳-JA分布在筛管、次生韧皮部的薄壁细胞和射线细胞、形成层区以及导管、次生木质部的薄壁细胞和射线细胞,其中,在韧皮部和木质部的射线细胞及筛管、导管壁周围分布最多。JA 在橡胶树内可快速通过韧皮部经由形成层径向运输到木质部,同时,JA 能通过木质部和韧皮部进行纵向长距离双向运输。     

Characterization of zein modified with a mild cross-linking agent

Zein, a predominant corn protein, is an alcohol-soluble protein extracted from corn and is an excellent film former. The characteristic brittleness of zein diminishes its usefulness as a film. It is well known that zein has a propensity for forming aggregates in solution. When zein molecules were cross-linked with 1-[3-dimethylaminopropyl]-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), it was found that the film-forming property was improved and the aggregation phenomenon in solution was suppressed. At the air/water interface, native zein forms brittle film with rough surface, whereas cross-linked zein forms rigid film with very smooth and even surface. Tensile strengths of the films were shown to be greatly increased by cross-linking. Objectives of this study are to determine the cross-linking mechanism of zein, the optimum reaction conditions, characteristics of the reaction product, and mechanical properties of cross-linked zein film. Through viscosity and dynamic light scattering, the cross-linking reaction was monitored. Optimum amount of EDC and NHS was determined to be 30 mg each per gram of zein. The cross-linking of zein with EDC and NHS seemed to be self-terminating because the cross-linking reaction did not proceed to precipitation.     

A method for the determination of chlorpropham residues in crisps and crisp frying oil

Summary Chlorpropham residues in potato crisps were determined by a method of analysis involving solvent extraction, alumina column clean-up and quantification of the chlorpropham by gas chromatography. The method has an overall recovery rate of 93.2% and the minimum detectable amount is equivalent to 0.035 mg kg¹. Commercial samples having an initial residues of 0.18 mg kg¹ in washed, peeled slices contained a residue of 0.45 mg kg¹ after frying; the frier oil contained 0.4 mg kg¹.     

Preparation,Identification, Molecular Docking Study and Protective Function on HUVECs of Novel ACE Inhibitory Peptides from Protein Hydrolysate of Skipjack Tuna Muscle

To prepare bioactive peptides with high angiotensin-I-converting enzyme (ACE)-inhibitory (ACEi) activity, Alcalase was selected from five kinds of protease for hydrolyzing Skipjack tuna (Katsuwonus pelamis) muscle, and its best hydrolysis conditions were optimized using single factor and response surface experiments. Then, the high ACEi protein hydrolysate (TMPH) of skipjack tuna muscle was prepared using Alcalase under the optimum conditions of enzyme dose 2.3%, enzymolysis temperature 56.2 °C, and pH 9.4, and its ACEi activity reached 72.71% at 1.0 mg/mL. Subsequently, six novel ACEi peptides were prepared from TMPH using ultrafiltration and chromatography methods and were identified as Ser-Pro (SP), Val-Asp-Arg-Tyr-Phe (VDRYF), Val-His-Gly-Val-Val (VHGVV), Tyr-Glu (YE), Phe-Glu-Met (FEM), and Phe-Trp-Arg-Val (FWRV), with molecular weights of 202.3, 698.9, 509.7, 310.4, 425.6, and 606.8 Da, respectively. SP and VDRYF displayed noticeable ACEi activity, with IC₅₀ values of 0.06 ± 0.01 and 0.28 ± 0.03 mg/mL, respectively. Molecular docking analysis illustrated that the high ACEi activity of SP and VDRYF was attributed to effective interaction with the active sites/pockets of ACE by hydrogen bonding, electrostatic force, and hydrophobic interaction. Furthermore, SP and VDRYF could significantly up-regulate nitric oxide (NO) production and down-regulate endothelin-1 (ET-1) secretion in HUVECs after 24 h treatment, but also abolish the negative effect of 0.5 μM norepinephrine (NE) on the generation of NO and ET-1. Therefore, ACEi peptides derived from skipjack tuna (K. pelamis) muscle, especially SP and VDRYF, are beneficial components for functional food against hypertension and cardiovascular diseases.     

硝酸镧对蒲葵幼苗耐寒性生理指标的影响

用0、100、200、300、400、500、600 mg/L硝酸镧溶液喷施蒲葵幼苗叶片作为处理，每天喷施1次，连续3 d。在20、5、12和10 ℃下分别处理24 h后测定叶片的丙二醛、可溶性糖和叶绿素含量以及叶绿素a/b的比值。结果表明：硝酸镧溶液处理的幼苗丙二醛含量都比对照低，而且随温度变化幅度小，而可溶性糖和叶绿素含量及叶绿素a/b比值均比对照高。硝酸镧处理可有效改善上述蒲葵幼苗的耐寒性生理指标，其中300 mg/L硝酸镧溶液对提高蒲葵幼苗的耐寒性生理指标具有显著的效应。     

硝酸镧处理对短穗鱼尾葵幼苗耐寒性的影响

为探明硝酸镧对短穗鱼尾葵(Caryotamitis)耐寒生理指标的影响,用4个不同浓度的硝酸镧溶液(0、100、350、500mg/L)喷施盆栽短穗鱼尾葵叶片,经变温处理后检测各项耐寒指标。结果表明,超氧化物歧化酶(SOD)、过氧化物酶(POD)、丙二醛(MDA)、脯氨酸、可溶性蛋白和叶绿素的含量表现出明显的浓度效应,而且25℃恢复后幼苗叶片的各项耐寒指标的模糊隶属函数值分别为0.41、0.46、0.70、0.50,其中350mg/L硝酸镧溶液处理对提高短穗鱼尾葵幼苗的耐寒性具有显著的效应。     

冬樱花扦插生根处理研究

采用不同质量百分数浓度的NAA、国光生根剂(20% NAA)、根旺(NAA、IBA各占1%)溶液浸渍冬樱花插穗15～30 s,进行扦插生根试验。结果表明：NAA＋IBA组合使用明显优于NAA单独使用,500 mg/kg根旺(NAA　250 mg/kg＋IBA　250　mg/kg)时冬樱花扦插生根率最高,达80.3%。此繁殖方法可以在冬樱花推广种植时应用。     

Blend films based on silk fibroin/hyaluronic acid

Protein and polysaccharide was the most important extracellular matrix in dermal tissue. In this study, Silk fibroin (SF) / hyaluronic acid (HA) blend films mimicking the dermal tissue components were prepared and investigated. The results indicated that HA and SF has a good miscibility, HA interfered with SF to form crystal structure. By using EDC as cross-linker, effective cross-linking function on SF and HA macromolecules was reacted, the water solubility of the blend films decreased obviously after being cross-linked by EDC. The existence of EDC could promote SF to form Silk I structure. L929 cells were seeded on these blend films and showed normal attachment morphology. Cell-matrix interactions established by newly formed extracellular matrix were observed after 5 days in culture. The MTT assay showed that cell proliferation on the SF/HA blend films were enhanced significantly compared with that on the SF and HA films. These new 2D SF/HA blend films provided a favorable microenvironment for the proliferation of L929 cells and hold a potential for dermal tissue regeneration.     

不同用量的钾镁肥在砖红壤中的交互效应

研究不同用量的钾镁肥在砖红壤中的相互作用规律,可为合理施用钾镁肥提供理论依据。供试土壤采自琼海市大路镇冬瓜种植地,在室内试验条件下,通过向砖红壤中加入不同用量的钾镁肥,每隔一段时间,测定土壤中钾镁的有效含量。结果表明,施用钾镁肥是提高土壤中速效钾和交换性镁含量的有效方法;在不施钾肥时,无论施用镁肥与否,对土壤中速效钾含量没有影响,当施钾量分别达到50、250 mg/kg时,施高镁肥（150 mg/kg）对钾有拮抗作用;在不施镁肥或施少量镁肥（30 mg/kg）时,无论施用钾肥与否,对土壤中交换性镁含量都没有影响,但当施高量镁肥时,钾对镁有拮抗作用,但随着培养时间的延长,拮抗作用减弱。