Isolation and partial characterization of the outer dense fibres and fibrous sheath from the sperm tail of a marsupial: the brushtail possum (Trichosurus vulpecula)

The flagellum of a mammalian spermatozoon consists of a central axoneme surrounded by two cytoskeletal structures, the outer dense fibres and the fibrous sheath, which may aid in sperm motility or stability. In this study the outer dense fibres and fibrous sheath were isolated and partially characterized in a marsupial species, the brushtail possum (Trichosurus vulpecula). Spermatozoa from the cauda epididymidis were decapitated by sonication, and the head and tail fractions were separated by centrifugation over a 20, 40 and 60% (w/v) sucrose density gradient. After confirming sperm tail purity by Nomarski microscopy, the tails were incubated in either SDS-dithiothreitol to isolate the outer dense fibres or urea-dithiothreitol to isolate the fibrous sheaths. Purified outer dense fibres and fibrous sheaths were solubilized in SDS and beta-mercaptoethanol and proteins were separated by one-dimensional PAGE. Coomassie blue staining showed that the outer dense fibres were composed of seven major proteins (molecular masses: 73, 58, 55, 54, 52, 41 and 16 kDa), and the fibrous sheath was composed of 12 major proteins (molecular masses: 106, 76, 66, 62, 55, 53, 52, 46, 40, 30, 28 and 16 kDa). A polyclonal antibody to the fibrous sheath proteins showed strong crossreactivity with those of fibrous sheath from spermatozoa of several other marsupial species, as well as those from laboratory rats. Subsequent western blotting identified the immunoreactive 76 and 62 kDa proteins from all species, thus indicating their high conservation between species. No crossreactivity of the fibrous sheath antibody to any other cytoskeletal structures, including the outer dense fibres, mid-piece fibre network or connecting laminae, or to the acrosome or underlying subacrosomal material, was evident, indicating that the fibrous sheath proteins are localized to this structure alone. Further work is in progress to determine the extent of homology of these proteins to those in eutherian mammals.     

应用抗黄曲霉毒素单链抗体检测酱油中黄曲霉毒素B1

利用本实验室制备的抗黄曲霉毒素B1的单链抗体(ScFv),通过棋盘实验确定了抗原抗体的最适工作浓度,在此之上根据间接竞争酶联免疫法(ELISA)绘制标准曲线,检测酱油中AFB1的含量;通过改变样品的盐浓度及pH来确定其对ELISA检测结果的影响。研究结果表明,利用ScFv检测黄曲霉毒素的最小检测值为0.10ng/mL,平均加标回收率在84%～109%之间,本文建立的ELISA方法在pH5～8,盐浓度小于10%时较稳定。本文建立的利用抗AFB1的ScFv检测黄曲霉毒素含量的方法方便快捷,稳定性较好,并且成本较低,适合于食品中黄曲霉毒素的检测。     

反应条件对黄曲霉毒素B1抗原-抗体结合的影响分析

目的 探索酶联免疫吸附测定(enzyme linked immunosorbent assay, ELISA)反应中反应条件对抗原(antigen, Ag)与抗体(antibody, Ab)结合的影响。方法 以黄曲霉毒素B1 (aflatoxin B1, AFB1)抗原抗体为主要研究对象, 通过分析ELISA中包被方法、包被液、离子浓度、pH、时间、温度、终止液等不同反应条件下的吸光度值(optical density, OD)和抑制率等指标, 了解反应过程中抗原与抗体结合的差异情况, 讨论反应条件对抗原-抗体结合的影响。结果 两步包被抗体法效价高、抑制率好, 包被液用pH 7.0、0.1 mol/L的磷酸氢二钠-磷酸二氢钾抑制率高, 封闭时间影响较小, 25℃反应更适合操作, 辣根过氧化物酶(horseradish peroxidase, HRP)标记抗原稀释液离子浓度0.020 mol/L pH=7.0时抗原-抗体结合最好, 用盐酸比硫酸作为终止液更稳定。结论 包被方法、包被液、离子浓度、pH、时间、温度、终止液等条件, 均是影响抗原-抗体结合的因素。     

不同前处理方法对粮油中黄曲霉毒素B1检测效果对比

考察3种前处理方法的净化效果,建立柱后光化学衍生-高效液相色谱法对粮食中黄曲霉毒素B1的检测方法.样品经甲醇+水(70∶30)提取,常规手动亲和柱净化、全自动亲和柱净化及免疫磁珠-全自动净化3种前处理净化,经Eclipse plus C18色谱柱分离,用64％水+18％甲醇+18％乙腈等度洗脱.优化色谱条件后,从加标回...     

Quantitative determination of aflatoxin B1 in chick liver

A method for the rapid and quantitative determination of aflatoxin B1 from small quantities of liver, around 1-2 g, is described. The extraction procedure involves acidification to pH 2 of the aqueous liver homogenates, extraction with chloroform: acetone and HPLC-fluorimetric detection after derivatization with trifluoroacetic acid. Quantitative recovery of aflatoxin B1 from chick liver was achieved and detection at levels of 0.2-1 ppb was proved feasible. The aflatoxin B1 concentration in chick liver after oral administration is also shown.     

高效液相色谱法测定粮谷中黄曲霉毒素B1的研究

建立了用三氯甲烷提取、硅胶小柱净化、三氟乙酸衍生,再以荧光检测器来测定粮谷中黄曲霉毒素B1的高效液相色谱方法.试验证明,该方法安全限量标准最低可达0.4 μg/kg,线性在2～50 μg/kg良好,平均回收率较高,具有准确度高、精密度好、安全限量低的特点.     

黄曲霉毒素B1降解菌株的筛选及鉴定

该研究以香豆素为唯一碳源,从豆类发酵食品、土壤、动物肠道及其内容物中筛选黄曲霉毒素B1（AFB1）降解菌株;然后通过复筛,从中筛选出AFB1降解能力较好的菌株,并对其降解作用与吸附作用进行区分;最后,通过形态观察、生理生化试验及分子生物学技术对其进行鉴定。结果表明,共筛选得到9株AFB1降解菌株,其中菌株YC2的AFB1降解能力最强,AFB1降解率达到90.7%,并确定菌株YC2通过降解作用去除AFB1。最后,菌株YC2被鉴定为枯草芽孢杆菌（Bacillus subtilis）。     

Excretion pattern of aflatoxin M1 in milk of goats fed a single dose of aflatoxin B1

The feedstuffs used in dairy animals must be able to give consumers confidence about the wholesomeness of milk with regard to aflatoxin contamination. The aim of this study was to determine the excretion patterns of aflatoxin M(1) (AFM1) in the milk of dairy goats fed a single dose of pure aflatoxin B(1) (AFB1), which can occasionally occur if feeds are infected by hot-spot growth of molds that produce aflatoxins. Five dairy goats in midlactation were administered 0.8 mg of AFB1 orally. Individual milk samples were collected for 84 h after AFB1 dosage. Aflatoxin M(1) was found in milk in the highest concentration. In all goats, AFM1 was not detected in milk before AFB1 administration, but was detected in the first milking following AFB1 administration. The excretion pattern of AFM1 concentration in milk was very similar in all goats even if the values of the concentration differed between animals. The peak values for AFM1 concentration in milk was observed in milk collected during the milking at 3 and 6h. After the peak, the AFM1 in milk disappeared with a trend that fitted well a monoexponential decreasing function, and the toxin was not detected after 84 h. Only about 0.17% of the amount of AFB1 administered was detected as AFM1 in milk, and about 50% of this was excreted in the first liter of milk yielded after AFB1 intake. Correct procedures to prevent growth of molds, and consequent AFB1 contamination, on the feedstuffs for lactating goats represent the key to providing consumers a guarantee that milk is not contaminated by AFM1.     

生物技术对黄曲霉毒素B1的脱除及机制研究进展

黄曲霉毒素是危害最大的真菌毒素之一,而黄曲霉毒素B₁(AFB₁)是黄曲霉毒素中毒性最大的一种,具有高毒性、致癌、致畸和致突变的作用,对动物和人类健康造成危害。为了有效脱除AFB₁,综述了近年来研究的具有解毒作用的微生物,介绍了采用生物技术脱除AFB₁的方法,重点探讨了生物脱除AFB₁机制。用于脱除AFB₁的菌株有芽孢杆菌、假单胞菌、乳酸菌、非产毒曲霉等,可采用单菌种发酵、多菌种协同发酵和菌-酶协同作用脱除AFB₁。生物脱除AFB₁机制主要为降解脱除和吸附脱除。可进一步筛选具有高优良能力的菌株以及更深层次地研究微生物脱毒的机制,探索微生物制剂对AFB₁的降解作用。     

时间分辨荧光免疫法检测食品中黄曲霉毒素B1的含量

目的建立高灵敏度的时间分辨免疫法检测黄曲霉毒素B_1的含量。方法通过双功能螯合剂异氰酸苄基二乙烯三胺四乙酸络合稀土元素Eu~(3+)标记抗AFB_1的单克隆抗体,以AFB_1-OVA为固相抗原,优化反应条件,建立直接竞争时间分辨免疫检测方法。结果优化条件后,方法灵敏度为0.02μg/L,IC_(50)为0.73μg/L,线性检测范围为0.01~30μg/L,大米、花生、黄豆和干果样品回收率在91%~104%之间,检测结果准确;与结构类似物AFB_2、AFG_1、AFG_2、AFM_1的交叉反应率分别为17.3%、4.49%、2.37%和0.73%。结论本方法灵敏度高、特异性强、检测快速,可以满足实际样品的检测需求。     

花生油生物精炼法去除黄曲霉毒素的研究

目前已经筛选出能够固态发酵花生粕降解其中黄曲霉毒素B1(AFB1)的微生物菌株,因此可以利用发酵液提取粗酶来降解花生油中的AFB1,并将这种酶反应引入到花生油的精炼工艺中.通过对比精炼工艺,将酶法脱胶(采用磷脂酶A1)与去毒反应相结合.对脱胶去毒过程中反应温度、NaOH添加量、去毒粗酶添加量、磷脂酶A1添加量、反应时间等进行单因素实验,并通过正交实验确定脱胶去毒实验优化工艺条件为:反应温度40℃,反应时间4h,NaOH添加量0.025％,磷脂酶A1添加量600 mg/kg.以添加AFB1为100 μg/kg的花生毛油为实验对象,取样10 g,在去毒粗酶添加量100 μL及优化条件下,AFB1去除率高达81％,去毒反应后花生油的脱胶效果也达到精炼要求(磷含量降至13 mg/kg).     

Detection and quantitation of aflatoxin B1 in orange juice by SOS-Chromotest

A new method for the detection and quantitation of aflatoxin B1 in liquids is described. The method is based on the SOS Chromotest, in which damage caused by aflatoxin B1 to the DNA of suitably engineered E. coli induces beta-galactosidase. Aflatoxin B1 developing in orange juice inoculated with spores of Aspergillus parasiticus is detectable equally well by TLC as by the SOS-Chromotest.     

Co-contamination of aflatoxin B1 and fumonisin B1 in food and human dietary exposure in three areas of China

Aflatoxins and fumonisins are ubiquitous foodborne toxicants and the co-occurrence of these mycotoxins in human foods represents a significant public health concern, which has been strongly associated with human aflatoxicosis, neural tube defects, as well as many types of primary cancers. In this study the co-contamination of aflatoxin B(1) (AFB(1)) and fumonisin B(1) (FB(1)) in food and human dietary exposure was investigated in residents of three different areas of China. A total of 209 food samples were measured for AFB(1) and FB(1). The median AFB(1) levels were 13.5, 2.3 and 1.3 μg kg(-1) and the median FB(1) levels were 2.6, 0.4 and 0.3 mg kg(-1) in corn samples collected from Huaian (a high-risk area for oesophageal cancer), Fusui (a high-risk area for liver cancer) and Huantai (a low-risk area for both oesophageal and liver cancers), respectively. The median level of AFB(1) in plant oil of Fusui was the highest (52.3 μg kg(-1)) among all food samples analysed. Co-contamination of these two mycotoxins was found in corn, rice and wheat flour. Based on measured food consumption data, the averaged daily dietary intake of AFB(1) was 0.397 μg (range = 0.269-1.218 μg) in residents of Huantai, 1.723 μg (0.224-49.772 μg) in Huaian, and 2.685 μg (1.006-14.534 μg) in Fusui. The averaged FB(1) daily dietary intake was 92.4 μg (range = 55.0-362.1 μg) for residents of Huantai, 460.0 μg (83.2-2894.5 μg) in Huaian, and 138.6 μg (30.0-10,541.6 μg) in Fusui. These data suggest that the co-exposure to AFB(1) and FB(1) in residents of rural China may contribute to the aetiology of human chronic diseases in high-risk areas.     

Co-contamination of aflatoxin B1 and fumonisin B1 in food and human dietary exposure in three areas of China

Aflatoxins and fumonisins are ubiquitous foodborne toxicants and the co-occurrence of these mycotoxins in human foods represents a significant public health concern, which has been strongly associated with human aflatoxicosis, neural tube defects, as well as many types of primary cancers. In this study the co-contamination of aflatoxin B₁ (AFB₁) and fumonisin B₁ (FB₁) in food and human dietary exposure was investigated in residents of three different areas of China. A total of 209 food samples were measured for AFB₁ and FB₁. The median AFB₁ levels were 13.5, 2.3 and 1.3?µg?kg^?1 and the median FB₁ levels were 2.6, 0.4 and 0.3?mg?kg^?1 in corn samples collected from Huaian (a high-risk area for oesophageal cancer), Fusui (a high-risk area for liver cancer) and Huantai (a low-risk area for both oesophageal and liver cancers), respectively. The median level of AFB₁ in plant oil of Fusui was the highest (52.3?µg?kg^?1) among all food samples analysed. Co-contamination of these two mycotoxins was found in corn, rice and wheat flour. Based on measured food consumption data, the averaged daily dietary intake of AFB₁ was 0.397?µg (range?=?0.269–1.218?µg) in residents of Huantai, 1.723?µg (0.224–49.772?µg) in Huaian, and 2.685?µg (1.006–14.534?µg) in Fusui. The averaged FB₁ daily dietary intake was 92.4?µg (range?=?55.0–362.1?µg) for residents of Huantai, 460.0?µg (83.2–2894.5?µg) in Huaian, and 138.6?µg (30.0–10,541.6?µg) in Fusui. These data suggest that the co-exposure to AFB₁ and FB₁ in residents of rural China may contribute to the aetiology of human chronic diseases in high-risk areas.     

2011-2012年食用植物油中黄曲霉毒素B1的调查

为调查我国市场食用植物油油中黄曲礞毒素B1是螽符合国家标准规定,本论义膈两年的时间,对988个传统烹调油样品、342个新兴功放类油样品中的黄曲霉毒素B1进行了检测。结果显示：2011年传统烹调油合格率为99．22％,新必功效类油样,铺及凋味汕合格宰为95．03％;2012年传统烹调油合格率为99．37％,新必功效类油样品及调味油合格半为96．90％。由此可见,食用油产品的合格牢征逐年提高。     

细菌降解黄曲霉毒素B1的研究进展

黄曲霉毒素B1是目前已发现的黄曲霉毒素中毒性最强的一种,其具有强肝毒性、高致突变性和高致畸性。广泛存在于农产品及饲料食品中,对人类健康存在严重威胁,同时对粮食和畜牧业造成严重的经济损失。细菌降解黄曲霉毒素B1是一种有效、安全和环保的解毒方法,该文通过对黄曲霉毒素B1降解的影响因素、细菌胞外酶和胞内酶等对黄曲霉毒素B1的降解机理以及黄曲霉毒素B1降解菌活性产物的应用研究等方面对细菌降解黄曲霉毒素B1进行了论述,并对细菌降解黄曲霉毒素B1应用前景进行展望,为以后更进一步研究提供较为全面的资料。     

抗黄曲霉毒素B1独特型纳米抗体的表达及复性

目的 快速制备大量具有生物活性的独特型纳米抗体F7。方法 构建pET22b-F7原核表达载体, 转化至大肠杆菌E. coli BL21(DE3)中进行诱导表达, 对诱导温度、诱导剂浓度和诱导时间进行优化。结果 独特型纳米抗体F7表达量有所增加但主要以包涵体形式存在。经过包涵体的溶解、复性获得了具有生物活性的抗AFB1独特型纳米抗体, ELISA证实复性后的蛋白依然存在对鼠源AFB1抗体的结合特性。结论 为后续抗体的性质研究和改造奠定了基础。