大豆(Glycine max(L.)Merr.)致敏蛋白Gly m Bd 30K检测方法及低致敏优异种质鉴定

Gly m Bd 30K蛋白是大豆中主要的免疫显性过敏原之一,会引起人和牲畜腹泻和肠道炎症等过敏反应.因此,发掘低Gly m Bd 30K蛋白含量优异种质对于培育优质大豆品种具有重要意义.为了获得致敏蛋白Gly m Bd 30K低含量的优异种质,根据Gly m Bd 30K蛋白的190-379aa多肽序列制备多克隆抗体...     

Calibration of prestained protein molecular weight standards for use in the "Western" or enzyme-linked immunoelectrotransfer blot techniques

Prestained protein molecular weight standards allow easy, direct visual location of electrophoretically transblotted lanes on nitrocellulose. They also provide a simple and accurate means for calibrating the molecular weights of resolved bands. Commercial prestained protein molecular weight standards, however, appear to have significantly different molecular weights from the original unstained proteins. We describe a calibration of these prestained molecular weight standards.     

大鼠仙台病毒ELISA、间接免疫荧光和免疫印迹三种检测方法比较

目的比较ELISA（enzyme-linked immunosorbent assay）、IFA（immuno-fluorescence assay）和WB（Western blot）三种方法在大鼠仙台病毒血清学检测中的差异。方法仙台病毒蛋白抗原经凝胶电泳分离转移后用于血清学检测的WB方法;使用IFA、ELISA方法对20份无菌大鼠、227份SPF大鼠以及63份清洁级大鼠送检血清样品进行检测,阳性及可疑样品用WB方法进行了验证。结果 20份无菌大鼠血清样品被3种方法检测为仙台病毒抗体阴性;SPF级大鼠样品被IFA方法判定为阴性,1.32%（3/227）被ELISA方法判定为阳性,其中有2/3被WB确认为阳性;ELISA、IFA和WB在清洁级大鼠样品中检出仙台病毒的阳性率分别为为18.12%、11.34%和15.87%。结论三种检测方法灵敏度从高到低依次为ELISA、WB和IFA。WB方法可作为IFA和ELISA难以确定结果的替代方法。     

中枢注射抗Orexin抗体对禁食大鼠摄食的抑制作用

目的:探讨中枢注射抗Orexin抗体对禁食大鼠摄食的抑制作用。方法:采用免疫组织化学法和蛋白质免疫印迹分析法,对Orexin抗体的特异性进行了检测,分析Orexin阳性神经元和阳性神经纤维在大脑中的分布。给予24 h禁食大鼠中枢注射抗Orexin抗体,计算其对大鼠食物摄入量的影响。结果:蛋白质免疫印迹分析显示,Orexin抗体能够检测到合成的Orexin-A。免疫组织化学分析显示,Orexin阳性神经元存在于外侧下丘脑区域和穹窿周核,Orexin阳性神经纤维大量投射至弓状核、下丘脑室周核和下丘脑室旁核、菱形丘脑核、丘脑室旁核、缰内侧核和纹质丘脑、中脑的中央灰质区、蓝斑核和中缝核、脑桥和髓质的网状结构、对疑核和迷走神经复合体。与注射羊血清相比,给予45μg/10μL抗Orexin抗体侧脑室注射则抑制了大鼠的食物摄入量(P0.05)。高剂量抗Orexin抗体能显著地抑制大鼠摄食,并且呈剂量依赖关系(P0.05)。结论:中枢注射抗Orexin抗体对禁食大鼠摄食具有抑制作用,并呈剂量依赖关系。     

A ferredoxin-type iron-sulfur protein gene,frx B,is expressed in the chloroplasts of tobacco and spinach

Previously, a ferredoxin-type iron-sulfur protein, frx B protein, was identified in a high-salt extract of the purified thylakoid membrane of Chlamydomonas reinhardtii, a unicellular green alga. Polyclonal antibody was raised against a synthetic pentadecameric peptide with an amino acid sequence corresponding to the highly conserved region of the putative frx B proteins of 3 land plants [21]. In this report, protein(s) reacting strongly and specifically with this antibody was detected in the equivalent high-salt extract prepared from purified chloroplast of spinach and tobacco. One strong reaction polypeptide band from tobacco chloroplast was purified from SDS-polyacrylamide gel and subjected to endoproteinase lys C digestion. The resulting polypeptides were separated by reversed-phase chromatography. N-terminal sequencing of 3 purified polypeptides revealed that the protein is encoded by the frxB gene identified from DNA sequence analysis.     

应用反向线点杂交技术鉴定临床常见曲霉属和毛霉目真菌

目的应用反向线点杂交技术(reverse line blot hybridization,RLB)快速鉴定临床常见的曲霉属和毛霉目真菌。方法收集我院真菌和真菌病研究中心保存的5种曲霉菌(烟曲霉、黄曲霉、黑曲霉、土曲霉、构巢曲霉)和7种毛霉目真菌(冻土毛霉菌、总状毛霉菌、卷枝毛霉菌、少根根霉、小孢根霉、微小根毛霉、伞状犁头霉),共计98株菌株。利用真菌通用引物ITS1和ITS4对菌株进行PCR扩增,用12个真菌种特异性探针与扩增后产物进行反向线点杂交。将RLB结果与真菌传统形态学鉴定结果、ITS区DNA测序结果进行比较。结果 RLB可以正确鉴定98株实验菌株,与形态学方法和ITS区测序方法鉴定结果100%一致,种特异性探针之间未见交叉杂交,显示出该方法的高度敏感性和特异性。8株阴性对照菌株(白念珠菌、茄病镰刀菌、尖端赛多孢、马尔尼菲青霉、疣状瓶霉、棒曲霉、日本曲霉以及雅致小克银汉霉),使用RLB方法无法鉴定。通过烟曲霉基因组DNA浓度10倍倍比稀释法验证RLB的敏感性为1.8×10-3 ng/μL。结论 RLB技术为实验室早期快速诊断、鉴定临床常见的曲霉属和毛霉目真菌提供参考。     

A new approach to monitor expression of aldo–keto reductase proteins in mouse tissues

The aldo–keto reductase (AKR) proteins catalyze reduction of diverse aldehydes and play detoxification roles in many organisms. Since many substrates are shared among AKR, it is generally accepted that these enzymes can functionally compensate each other in response to oxidative stress. Their overall abundances are the important factor that partially reflects the capacity of antioxidant and detoxification in tissues. In this study, the strategy was proposed for generation of Pan‐AKR antibodies to recognize most AKR proteins in mouse tissues. Derived from bioinformatic analysis, several consensus peptides with different potential antigenicities were synthesized, conjugated to hemocyanin from keyhole limpets and further delivered to rabbits to generate polyclonal antibodies. Three Pan‐AKR antibodies exhibited the immune specificities and immune sensitivities, Pan‐AKR‐P1 for AKR1B and AKR1C, Pan‐AKR‐P3 for AKR1C and Pan‐AKR‐P4 for all the AKR proteins. Pan‐AKR‐P4 antibody was employed to 2‐DE Western blot to examine the AKR abundances in mouse liver and kidney, resulting in seven immune‐reactive spots from each tissue. Protein identification with MS revealed that most immune‐positive spots were the members of AKR superfamily. Furthermore, Pan‐AKR‐P4 antibody was implemented to compare the different abundances of the AKR proteins in liver and kidney between normal and diabetic mice, suggesting that diabetes did cause some abnormal changes in the AKR protein abundances.     

Incidence of Vibrio vulnificus in estuarine waters of the south Texas Coastal Bend region

Aims: To determine the occurrence of the human pathogen, Vibrio vulnificus, in south Texas coastal waters. Methods and Results: Coastal waters were sampled monthly between August 2006 and July 2007. Water temperature, dissolved oxygen, pH, salinity, conductivity and turbidity were measured during each sampling event. Culture‐based techniques utilizing Vibrio vulnificus agar (VVA) and membrane‐Enterococcus indoxyl‐β‐d ‐glucoside agar (mEI) were used to assess the occurrence and levels of V. vulnificus and the faecal contamination indicator group, enterococci, respectively. Vibrio vulnificus isolates were confirmed using colony‐blot hybridization with the species‐specific VVAP probe. Vibrio vulnificus was isolated at all sites throughout the year even when the water temperature dropped to 9·71°C. Significant correlations were found between concentrations of V. vulnificus and the abiotic factors, water temperature (P = 0·002) and dissolved oxygen (P = 0·028), as well as between concentrations of V. vulnificus and enterococci (P < 0·001). Conclusions: This study demonstrated the year‐round presence of V. vulnificus in coastal waters of south Texas. Significance and Impact of the Study: These findings indicate that the potential for human exposure to the pathogen, V. vulnificus, exists throughout the year. It also suggests that routinely monitored data might be used to predict the occurrence of the pathogen.