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.     

IgM型单克隆抗体之scFv库的建立与筛选

从一个抗脂磷酸聚糖IgM型单克隆抗体细胞的mRNA中经RT—PCR克隆重链可变区（VH）和轻链可变区（VL）,然后将VH、Linker和VL连接成seFv,并克隆到载体pCANTAB-5E,转Escherichia coli TG1感受态细胞,构建了单链抗体库。通过辅助噬菌体M13KO7感染后,使单链抗体展示在噬菌体衣壳蛋白pⅢ的N端,得到噬菌体展示的抗体库。将抗体库用于“淘洗-富集-扩增”3轮以后,得到针对脂磷酸聚糖特异性的噬菌体抗体。测序结果表明,该scFv的VH基因序列全长390bp,编码127个氨基酸;VL基因序列全长341bp,编码113个氨基酸。二者均符合小鼠免疫球蛋白可变区基因特征,含有4个框架区（FR）、3个抗原互补决定区（CDR）及抗体特征性的2个半胱氨酸残基。     

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。     

黄曲霉毒素B1标准替代物的制备及其在花生样品检测中的应用

以抗黄曲霉毒素B1（AFB1）单克隆抗体的F(ab’)2 片断为抗原免疫兔子,得到AFB1抗独特型抗体。经过酶联免疫吸附法（ELISA）条件的优化,建立AFB1抗独特型抗体最佳竞争抑制曲线。该曲线与AFB1竞争抑制曲线相比较可知,AFB1抗独特型抗体与AFB1之间存在指数增长关系,且相关性系数R为0.999 8。以AFB1抗独特型抗体浓度与其相应的抑制率作标准曲线,ELISA法测定花生中AFB1添加回收率,范围在90.4%~100.2%之间。综上,AFB1抗独特型抗体可以作为AFB1无毒替代标准品,为黄曲霉毒素检测无毒化提供了广阔的应用前景。     

纳米抗体特性及其在农产品真菌毒素检测中的应用

纳米抗体是由骆驼科动物产生的一类天然缺失轻链的重链抗体可变区.相比较于传统单克隆抗体和多克隆抗体,其具有体积小、热稳定性强、耐有机溶剂、折叠性可逆、易于表达、高亲和力、可特异性识别独特表位等天然优势.因此,纳米抗体在生物和农业等研究领域备受关注.本文综述了纳米抗体的结构、生化特性以及在农产品真菌毒素检测中的研究进展,并...     

抗黄曲霉毒素合成中关键蛋白AFLP(OmtA)的单克隆抗体制备与鉴定

AFLP(Omt A)蛋白是黄曲霉毒素合成晚期的关键酶。用大肠杆菌表达的重组SUMO-AFLP融合蛋白作为免疫原,免疫Balb/c小鼠,选取小鼠阳性血清的脾细胞与SP2/0骨髓瘤细胞融合,用间接ELISA和有限稀释法进行单克隆杂交瘤细胞的筛选,筛选出可以稳定分泌抗AFLP单克隆抗体的杂交瘤细胞株。再用阳性细胞株制备腹水抗体,经辛酸-硫酸铵纯化抗体后,用间接ELISA、Western杂交等方法对抗体的特性进行鉴定。筛选出1株能稳定分泌抗AFLP单克隆抗体的细胞株3B4 3B10,亚类鉴定单抗为Ig G2a。间接ELISA法测定该细胞腹水抗体的效价为1∶256 000,Western杂交结果显示抗AFLP单克隆抗体能特异性识别AFLP重组蛋白及黄曲霉菌内的天然AFLP蛋白。用1μg·m L~(-1)单克隆抗体检测AFLP蛋白,其线性检测范围为1.259~57.335ng·m L~(-1),检测限为0.851ng·m L~(-1),R~=0.998 3。本文成功制备抗黄曲霉毒素合成中天然AFLP蛋白的单克隆抗体,特异性和灵敏度均较高,为进一步研究AFLP的表达及功能机制奠定了基础。     

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.     

聚丙烯酰胺固相微球与黄曲霉毒素B1抗体偶联条件的研究

以表面带羧基的聚丙烯酰胺固相微球为载体,通过碳二亚胺(EDC)活化,共价结合兔黄曲霉毒素B1抗体.研究结果表明最适偶联pH值为6.0～6.5,最适反应时间为17～20h,最佳EDC用量为5～15mg/10mg微球.抗体的最大偶联效率达60.40%,为利用聚丙烯酰胺微球建立黄曲霉毒素快速检测技术奠定了基础.     

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。     

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。     

Diallel analysis of aflatoxin accumulation in maize

Since its discovery in numerous feedstuffs, aflatoxin, a carcinogenic compound produced by the fungus Aspergillus flavus Link ex Fries, has caused much concern among consumers and producers alike. This toxin poses a serious economic threat to maize (Zea mays L.) producers of the southeastern and midwestern regions of the United States. Efforts to identify maize germplasm that is resistant to aflatoxin accumulation and to investigate the genetic basis of this resistance have been undertaken at numerous research institutions. The objectives of this study were to (1) evaluate aflatoxin accumulation in grain harvested from maize inbred lines and a diallel cross among these lines, (2) determine the importance of general and specific combining abilities in inheritance of resistance to aflatoxin accumulation, and (3) estimate general and specific combining ability effects associated with resistance to aflatoxin accumulation in the inbred lines and crosses among them. Eight inbred lines and a diallel cross of the maize lines were inoculated with an A. flavus spore suspension 12–14 d after silk emergence. Following harvest, aflatoxin content was determined from samples of grain. Statistical analyses performed using SAS general linear models (GLM) and DIALLEL-SAS indicated that general and specific combining ability were significant sources of variation in the inheritance of resistance to aflatoxin accumulation. The inbred line Mp313E, which was developed and released as a source of resistance to aflatoxin contamination, had significantly lower aflatoxin accumulation than other lines. Mo18W exhibited excellent general combining ability for reduced aflatoxin accumulation when crossed with the other lines. Both Mp313E and Mo18W could be useful in breeding programs to develop aflatoxin-resistant maize hybrids. Mp339, SC212M, and Ab24E demonstrated aflatoxin susceptibility as both inbreds and in single crosses.     

Production of monoclonal antibodies against AFLM (Ver-1), a middle key protein involved in aflatoxin biosynthesis

Aflatoxins are potent carcinogens, mutagens and teratogens, and are harmful to both humans and animals. As many as 30 genes are involved in aflatoxin biosynthesis. Among them, aflM (ver-1) gene was predicted to encode a 28-kDa NADPH-dependent ketoreductase (AFLM), which catalyzed middle enzymatic steps in aflatoxin biosynthetic pathway. AFLM (Ver-1) was proved to be necessary for conversion of versicolorin A (VERA) to demethylsterigmatocystin (DMST) in aflatoxin B₁ (AFB₁) biosynthesis. For these reasons, aflM gene was cloned and specific monoclonal antibodies for AFLM was developed to better define potential pathways of AFLM involved in AFB₁ biosynthesis. Monoclonal antibodies 11B2-1D7 and 3G5-4E7 were successfully screened out by immunizing mouse. Immunoblot analysis revealed that both had high sensitivity and specificity to identify native AFLM protein in A. flavus with detection limit of 11 ​ng/mL and 8 ​ng/mL respectively. These results showed that it was suitable for quantitative detection of AFLM in A. flavus isolate. Further investigation revealed that aflatoxin accumulations of various A. flavus were not dependent on AFLM biosynthesis. Overall, this is the first report for development for AFLM monoclonal antibody development and application in A. flavus quantitative detection.     

粳稻大剑叶角资源的发现及剑叶角度的遗传分析与QTL定位

 利用粳稻保持系863B（P1）与A7444（P2）进行配组,构建了P1、P2、F1、B1(F1/P1)、B2(F2/P2)和F2 6个世代,并对剑叶角度进行遗传分析。调查了P1与P2及BC1F1世代141个单株SSR标记基因型和剑叶角度,构建该组合的SSR标记连锁图谱并定位剑叶角度的QTL。该连锁图谱由79个多态位点构成,全长441.6 cM,相邻标记的平均图距为5.6 cM。主基因加多基因的遗传模型分析结果表明,剑叶角度受2对主基因+多基因控制,以主基因遗传为主。单标记分析显示有15个标记与剑叶角度呈极显著相关。利用两种分析软件WinQTLCart 2.5和QTL Network 2.0共同检测到2个控制剑叶角度的QTL(qFLA2、qFLA8)。qFLA2位于RM300-RM145区间,qFLA8位于RM6215-RM8265区间,这两个QTL增效等位基因都来自A7444。     

Inheritance and QTL Mapping of Salt Tolerance in Rice

An F2 population derived from the cross between Jiucaiqing (japonica) and IR36 (indica) was used to analyze the inheritance of salt tolerance in rice by genetic model of major-genes plus polygenes, and to map the corresponding QTLs by SSR molecular markers. Rice plants of P1, P2, F1 and F2 at 5- to 6- leaf stage were treated under 140 mmol/L NaCI for 10 days. Three indices representing the ability of salt tolerance of rice seedlings were measured, including salt tolerance rating (STR), Na^ /K^ ratio in roots and dry matter weight of shoots (DWS). STR, Na^ /K^ and DWS were all controlled by two major genes with modification by polygenes. Heritability of these traits from major genes was 17.8, 53.3 and 52.3%, respectively. The linkage map constructed by 62 SSR molecular markers covered a total length of about 1 142 cM. There were three QTLs detected for STR located on chromosome 1, 5 and 9, two QTLs for DWS on chromosomes 8 and 9, and two QTLs for Na^ /K^ on chromosomes 2 and 6, one on each chromosome respectively. Single QTL accounted for 6.7 to 19.3% of phenotypic variation. Identification method of salt tolerance in rice and breeding of rice varieties with salt tolerance based on molecular markers assisted selection had been discussed.     

水稻品种资源91 1A②对稻瘿蚊的抗性鉴定及其遗传分析

 以抗虫品种资源91 1A②为父本,分别与粳桂、TN1、W1263（Gm1）、 IET2911（Gm2）、BG404 1（gm3）、OB677（Gm4）、ARC5984（Gm5）和多抗1号（Gm6）（母本）进行杂交、测交、自交,获得F1、BC1F1、F2群体,对亲本和各个群体进行稻瘿蚊的抗性鉴定,研究91 1A②的抗性基因和遗传规律。结果表明,91 1A②及其与其他品种杂交获得的F1都抗中国稻瘿蚊生物型Ⅳ型,杂交获得的BC1F1和F2对中国稻瘿蚊生物型Ⅳ型的抗虫和感虫植株分离比经χ2测验分别符合1∶3和9∶7,说明91 1A②对中国稻瘿蚊生物型Ⅳ型的抗性受2对显性基因控制。91 1A②所含的2对抗性基因与已知的6个抗性基因Gm1、Gm2、gm3、Gm4、Gm5和Gm6不等位,与其他5个抗性基因Gm7、Gm8、Gm9、Gm10、Gm11(t)也不等位。该抗性基因可能是2对新的抗性基因。     

Ear rot,aflatoxin accumulation,and fungal biomass in maize after inoculation with Aspergillus flavus

Aflatoxin, a toxin produced by the fungus Aspergillus flavus Link: Fries, occurs naturally in maize (Zea mays L.). Aflatoxin is a potent human carcinogen and is also toxic to livestock, pets, and wildlife. When contaminated with aflatoxin, the value of maize grain is markedly reduced. This investigation was conducted to compare ear rot, aflatoxin accumulation, and fungal biomass in maize single crosses with varying degrees of resistance to aflatoxin accumulation and to determine the relative importance of general combining ability (GCA) and specific combining ability (SCA) in the inheritance of resistance to ear rot, aflatoxin accumulation, and fungal biomass. Eight germplasm lines with different levels of resistance to aflatoxin accumulation were used as parents of a diallel cross. The cross was evaluated for visible ear rot, aflatoxin accumulation, and A. flavus infection in the grain. A. flavus infection was determined by quantitative real-time polymerase chain reaction (qRT-PCR) assays. Both GCA and SCA were significant sources of variation in the inheritance of the three traits although GCA accounted for a greater portion of the variation among single crosses. The interactions of GCA and SCA with years were highly significant for aflatoxin accumulation, but not significant for A. flavus infection. Estimates of GCA effects were highly significant for both reduced A. flavus infection and reduced aflatoxin accumulation for Mp313E, Mp715, and Mp717. Conversely, GCA effects associated with GA209 were significant for reduced levels of A. flavus infection and ear rot, but high levels of aflatoxin accumulation. Mp313E, Mp715, and Mp717 should be useful in breeding programs targeting both reduced levels of fungal infection and aflatoxin accumulation.     

Diallel analysis for aflatoxin accumulation and fall armyworm leaf-feeding damage in maize

Two major impediments to profitable maize, Zea mays L., production in the southern United States are from feeding by fall armyworm, Spodoptera frugiperda (J.E. Smith), and losses from the production and accumulation of aflatoxin in maize grain. A diallel cross was produced by making all possible crosses among five germplasm lines developed as sources of resistance to fall armyworm leaf feeding and five lines developed as sources of resistance to aflatoxin accumulation. For resistance to both leaf feeding and aflatoxin accumulation, general combining ability (GCA) was a significant source of variation. Specific combining ability (SCA) was significant for fall armyworm feeding only. Estimates of GCA effects for reduced aflatoxin accumulation were significant for Mp715 and Mp719, two lines selected for resistance to aflatoxin accumulation. The GCA effects for reduced fall armyworm damage were significant for all five lines selected for fall armyworm resistance: Mp707, Mp708, Mp713, Mp714, and Mp716.     

Cyclopeptide Derivatives from the Sponge-Derived Fungus Acremonium persicinum F10

Cyclopeptides usually play a pivotal role, either in the viability or virulence of fungi. Two types of cyclopeptides, six new hydroxamate siderophore cyclohexapeptides (1–6), including acremonpeptides E and F, and their complexes with aluminum and ferric ions; one new cyclic pentapeptolide, aselacin D (9); together with a known compound, aselacin C (10), were isolated and characterized from the sponge-derived fungus Acremonium persicinum F10. In addition, two new siderophore analogues chelating gallium ions (Ga³⁺), Ga (III)-acremonpeptide E (7) and Ga (III)-acremonpeptide F (8), using isolated acremonpeptides E and F, were prepared. The planar structures of 1–10 were elucidated by HRESIMS and (1D and 2D) NMR. The absolute configurations of amino acids were determined by means of the advanced Marfey’s method and X-ray single-crystal diffraction analysis. X-ray fluorescence (XRF) spectrometer was performed to disclose the elements of compound 1, indicating the existence of aluminum (Al). Al (III)-acremonpeptides E (1), Ga (III)-acremonpeptides E (5), Al (III)-acremonpeptide F (7), and Ga (III)-acremonpeptide F (8) displayed high in vitro anti-fungal activities, which are comparable to amphotericin B, against Aspergillus fumigatus and Aspergillus niger.     

利用重组近交系群体检测花生青枯病抗性SSR标记

用抗青枯病花生品种远杂9102与感病品种Chico杂交，从F2起用单粒传法构建了花生重组近交系群体（RIL）F6和F7。采用354对SSR引物对重组近交系F6群体的基因组DNA鉴定，获得多态性标记45个。结合重组近交系群体F6和F7青枯病抗性鉴定结果，应用相关软件统计分析，构建了栽培种花生部分遗传连锁图。图谱总长度为603.9cM，含29个标记（28个SSR标记和1个表型标记）的8个连锁群，还有17个独立的SSR标记；获得了与青枯病抗性相关的SSR标记2个（7G02和PM137），位于该图谱的第1连锁群上，与青枯病抗性基因间的遗传距离为10.9cM和13.8cM，并且位于抗性基因的两侧，两标记间的距离为23.7cM。