马立克氏病病毒MEQ蛋白对MDV增殖影响的研究

在马立克氏病病毒MDV致病机理的研究中，弄清致病基因meq与病毒增殖之间的关系及其分子机制是十分重要的基础，以重组反转录病毒（RCAS-meq)感染鸡胚成纤维细胞（CEF），使源于MDV强毒参考株（vMDV)GA株的meq基因表达于宿主细胞内，然后再用GA株感染这些细胞。通过利用MDV强毒株特异的抗pp38单克隆抗体所进行的“黑斑”试验以确定MDV的增殖水平，并与未接种RCAS-meq的CEF进行比较。研究结果发现，细胞内表达的meq基国产物可促进GA株于体外培养细胞中的感染与增殖（病毒斑数增多）。根据试验的结果，作者认为meq基因在感染细胞内的表达水平是MDV增殖以及进而能致病、致瘤的分子基础。     

马立克氏病研究的最新进展

文章对马立克氏病在病原的特性和致病机理、流行病学、诊断技术及防制方法等方面的研究进展进行了全面的概述，并提出了作者的一些见解和观点。     

马立克氏病病毒CVI988株病毒囊膜糖蛋白B启动子的克隆及活性分析

为了选择适宜的启动子调控外源基因的表达,以改善马立克氏病毒对载体的重组病毒的免疫保护力,克隆了马立克氏病病毒(MDV)CVI988株病毒囊膜糖蛋白B(gB)启动子,通过测序分析发现克隆的gB启动子与GenBank上发表的MDVGA株序列的同源性为99.5%。分别以β-半乳糖苷酶(β-galactosidase)和虫荧光素酶(luciferase)为报告基因,构建pSK-gB-LacZ和pgB-Luc真核表达载体。将pSK-gB-LacZ转染的中国地鼠卵巢(Chinesehamsterovary,CHO)细胞进行染色,出现蓝色,说明在CHO细胞中,MDVgB启动子可以有效地调控lacZ基因的转录。在CEF细胞中,利用虫荧光素酶系统将gB启动子与SV40及hCMV启动子进行活性比较,发现hCMV启动子的活性为gB启动子的31倍,SV40启动子为gB启动子的27倍。     

鸡抗马立克氏病遗传机制的研究进展

鸡的马立克氏病(MD)遗传抗性是由多个基因控制着,其中主要组织相容性复合体(MHC)基因是最确定的抗性影响因素.遗传抗病性研究根据病毒的生活史可分为抗感染和抗发病两个阶段,鸡抗MD的遗传机制研究目前主要集中在抗发病阶段,主要包括抗性基因的选择、抗性基因的表达水平、抗病性的遗传力.文章对上述与鸡马立克氏病遗传抗性相关的各个因素的研究进展进行了综述.     

鸡马立克氏病病毒(MDV)B抗原基因在烟草中的初步表达

为探索利用转基因植物产鸡马立氏病疫苗的新途径,将马立克氏病病毒（Ｍａｒｅｋ’ｓｄｉｓｅａｓｅｖｉｒｕｓ,ＭＤＶ）Ｂ抗原基因的两个片段,（Ｉ３和Ｋ３）整合连妆到相应的双元载体上,构建成带有ＮｐｔⅡ和Ｇｕｓ基因的ＭＤＶ Ｂ抗原基因的植物表达载体ｐＡｒｔ６９Ｉ３Ｋ３。     

广西霞烟鸡抗马立克氏病选育报告

鸡的马立克氏病(MD)抗病育种是防制 MD 的一个新方向。经三年多的研究,采用马立克氏病病毒(MDV)强毒株攻击霞烟鸡1日龄雏鸡,选留幸存者进行繁殖,其后代未经MD 疫苗接种,以自然感染进行选择,并结合后裔攻毒测定法进行抗 MD 的选育。至三世代时,鸡抗 MD 的能力已大为提高。三世代雏鸡1日龄攻毒后,10周龄时 MD 总保护率为74%,与对照组的35%有非常显著的差异(P<0.001),其中自然死亡鸡的 MD 肿瘤阳性率为16%,与零世代的48.1%有非常显著的差异(P<0.001).与对照组的37.5%亦有显著差异(P<0.05);1日龄经火鸡疱疹病毒(HVT)疫苗免疫,14日龄以 MDV 强毒攻毒后,三世代10周龄时对 MD总保护率为96.9%,而对照组为87%。抗病核心群目前已有基本种鸡500羽。     

表达GFP和gpt基因的重组CVI988病毒的构建

以马立克氏病病毒（MDV）CVI988株基因组为模板,利用PCR技术扩增出约2.7和3.0 kb的基因片段,将上述片段同时插入pUC19中,获得约5.5 kb MDV同源重组臂;以该基因片段的US2区的BglⅡ为插入位点,分别插入基因表达盒CMV-gpt-ployA和CMV-gfp-polyA,构建转移载体pUS-gpt-GFP,将该载体瞬时转染CHO细胞,在荧光显微镜下,可以观察到绿色荧光蛋白的表达;将该转移载体转染已用MDV CVI998株感染的次代CEF细胞,利用MX-HAT培养基筛选重组病毒,并用荧光显微镜挑选表达绿色荧光蛋白的蚀斑,结果获得重组病毒rMDVgptGFP。通过PCR检测和病毒生长测定,证明重组病毒获得纯化。     

Fusion Expression and Purification of Marek’s disease virus Tegument Protein VP16 and Its Antibody Preparation

马立克氏病病毒(Marek’s disease virus ,MDV)UL48基因编码的蛋白与单纯疱疹病毒1型(Herpes simplex virus ,HSV)衣壳蛋白VP16为同源物，将其克隆入pET-32a载体中，获得pET-VP16, 转化Escherichia coli BL21感受态细胞，经IPTG诱导表达，SDS-PAGE电泳显示:融合蛋白获得可溶性表达，表达蛋白的相对分子量约为67 kD；将表达的融合蛋白过His·Bind柱，获得纯化的蛋白，将该蛋白免疫家兔，制备多克隆抗体。通过间接ELISA和Western blot鉴定制备的多克隆抗体的效价和特异性，结果表明，该抗体具有较高的特异性，间接ELISA效价大于2×10－5。     

基因芯片技术检测5种马病毒的研究

通过分子克隆技术获得马疱疹病毒1型(Equine herpesvirus 1, EHV1)、马动脉炎病毒(Equine arteritis virus , EAV)、马流感病毒(Equine influenza virus, EIV)、马传染性贫血病毒(Equine infectious anaemia virus , EIAV)和东部马脑脊髓炎病毒(Eastern equine encephalomyelitis virus, EEEV)等5种病毒各一段高度保守的特异性基因片段，用芯片点样仪逐点分配到处理过的玻片上，制备成检测芯片。提取样品中的RNA，进行反转录和荧光标记后滴加到芯片上进行特异性杂交，对杂交结果进行扫描检测和计算机软件分析。结果显示，制备的基因芯片可同时检测和鉴别上述5种病毒，可检测到阳性杂交信号的最高稀释度为10-6的病毒液，约25个病毒DNA拷贝，但其它病毒材料未见红色荧光信号，证明了本方法的特异性。在进口马的隔离检疫期间，采集马鼻肺炎、马动脉炎中和抗体阳性但病毒分离阴性马匹的白细胞悬液，分别在EHV1和EAV位点处可检测到阳性杂交信号。证明基因芯片技术不但快速、准确和敏感，而且可同时进行多种病毒的检测。     

含绿色荧光蛋白基因与强毒部分糖蛋白B基因的马立克病毒CVI988株转移载体的构建

提取马立克病毒病毒疫苗毒株CVI988／Rispens感染的鸡（Gallus domesticus)胚成纤维细胞（CEF）的总DNA为模板，利用PCR技术扩增出病毒生长非必要的US2基因（约2.0kb)，将其克隆入T－easy载体获得载体pGUS2。用Eco R I和Bam H I消化pUR-MDVEgB质粒，回收1.8kb包含糖蛋白B（gB)基因主要抗原位点片段，插入pEGFP-C1质粒多克隆位点，构建了在CMV启动子和增强子控制下的含GFP及部分gB基因的表达盒。将此表达盒克隆入pGUS2载体US2基因中，成功地构建了含GFP及部分gB基因的转移质粒载体pEGFPgB。为其与CVI988毒株共转染CEF可获得表达GFP及强毒部分gB蛋白重组CVI988疫苗毒株打下基础。     

表达鸡传染性法氏囊病病毒VP2重组鸡痘病毒的构建

含ＩＢＤＶ保护性抗原ＶＰ２的质粒,以ＳｐｈＩ消化,然后用Ｔ４噬菌体ＤＮＡ聚合酶将末端补平,产物纯化后再以ＳａｌＩ消化一次,经再次纯化后与ＳｍａＩ和ＳａｌＩ分步酶切的鸡痘病毒插入载体ｐＦＧ１１７５－１相连,使唤基因顺向插入到载体Ｐ７５启动子下游,得到含ＩＢＤ ＶＰ２基因的重组插入载体ｐＦＧ ＶＰ２。     

新城疫病毒和减蛋综合征病毒同鸭胚增殖的研究

鸭源新城疫病毒 ( NDV D10 )和减蛋综合征病毒 ( EDSV)可以在鸭胚中同时良好增殖 ,两种病毒的血凝价分别与单独接种时一致 ;同胚增殖病毒对鸭胚或 SPF鸡胚的感染能力和致病性分别与单独接种组无显著差异 ;利用同鸭胚增殖病毒的尿囊液制备的二联油乳剂灭活疫苗接种免疫鸡可以分别抵抗 NDV强毒和 EDSV强毒的攻击     

辣椒病毒病病原种类检测初报

针对甘肃省农业科学院蔬菜研究所辣椒组育苗棚、试验地以及近郊辣椒种植田病毒病发病严重问题,开展了病毒病病原种类鉴定,并比较了双抗体夹心酶联免疫吸附法(DAS-ELISA)和简易试纸条对同一种病毒的检测结果。结果检测出了TMV、PVV和PVS 3种病毒,其中TMV为优势种群,侵染率高达66.7%,未发现CMV侵染。试验结果表明,DAS-ELISA法和简易试纸条法对同一种病毒检测结果基本一致。     

Disease and injury among participants in the Agricultural Health Study

The Agricultural Health Study (www.aghealth.org) is a cohort of 89,658 pesticide applicators and their spouses from Iowa and North Carolina assembled between 1993 and 1997 to evaluate riskfactorsfor disease in ruralfarm populations. This prospective study is just now reaching sufficient maturity for analysis of many disease endpoints. Nonetheless, several analyses have already provided interesting and important leads regarding disease patterns in agricultural populations and etiologic clues for the general population. Compared to the mortality experience of the general population in the two states (adjusted for race, gender, age and calendar time), the cohort experienced a very low mortality rate overall and for many specific causes and a low rate of overall cancer incidence. A few cancers, however, appear elevated, including multiple myeloma and cancers of the lip, gallbladder, ovary, prostate, and thyroid, but numbers are small for many cancers. A study of prostate cancer found associations with exposure to several pesticides, particularly among individuals with a family history of prostate cancer. Links to pesticides and other agricultural factors have been found for injuries, retinal degeneration, and respiratory wheeze. Methodological studies have determined that information collected by interview is unbiased and reliable. A third round of interviews scheduled to begin in 2005 will collect additional information on agricultural exposures and health outcomes. The study can provide data to address many health issues in the agricultural community. The study investigators welcome collaboration with interested scientists.     

Predicting Buckwheat Flavonoids Bioavailability in Different Food Matrices Under In Vitro Simulated Human Digestion

The digestibility and bioaccessibility of major flavonoids (rutin, quercetin, and isoquercitrin) in various buckwheat food matrices were evaluated as a function of the rutin levels using an in vitro simulated digestion model. Food matrices were unprocessed samples (buckwheat flour [BF], flavonoid extract [FE], rutin‐enhanced flavonoid extract [REFE], and pure rutin) and processed samples (cakes with BF, FE, and REFE and a rutin‐spiked cake). FE showed the highest digestibility out of all the unprocessed samples (FE > REFE > BF > rutin), whereas BF exhibited the highest bioaccessibility (BF > FE > REFE = rutin). Moreover, the processed samples improved their flavonoid bioaccessibility upon baking. Thus, unprocessed FE is a good source for highly bioavailable flavonoids; moreover, baking exerts a positive effect on flavonoid digestibility and bioaccessibility. Because flavonoids can be further fermented by microorganisms in the large intestine into various metabolites, determining the digestibility and bioaccessibility of various flavonoids is useful for predicting flavonoid bioavailability in buckwheat and buckwheat‐based food products.     

Interaction among phenols in food fortification: negative synergism on antioxidant capacity

In this work, a study about the consequences of the interaction among phenol compounds on antioxidant capacity is proposed. The antiradical activity evolution of an ethanol solution containing a mixture of three monophenols (catechin, resveratrol, and quercetin) was compared with the trend followed by each single phenol at three different temperatures (22, 37, and 60 degrees C). An initial increase and a following decrease in antioxidant activity were observed for all solutions at the three temperatures. The lower antiradical activity values of the mixture in comparison to the controls during the entire period of storage clearly showed that interaction among these polyphenols promotes a negative synergistic effect on this property.     

In Vitro Starch Digestibility of Tortillas Elaborated by Different Masa Preparation Procedures

Starch digestibility was evaluated in freshly prepared tortillas elaborated from masa obtained from different procedures (laboratory‐made masa, commercial masa, and nixtamalized corn flour) and from laboratory‐made masa with added commercial hydrocolloid, and stored for 24, 48, and 74 hr. Tortillas prepared with commercial masa had the highest available starch (AS) content and the commercial tortillas had the lowest, showing a decrease in AS content when storage time increased. Tortilla of commercial masa showed the lowest resistant starch (RS) content that agrees with the AS measured. However, tortilla of laboratory‐made masa presented the highest AS and RS contents. RS increased with storage time, a pattern that is related to the starch retrogradation phenomenon observed when retrograded resistant starch (RRS) was quantified. Commercial tortillas showed predicted glycemic index (pGI) values of 62–75% using a chewing/dialysis procedure (semi in vitro method). Index values were lower than those determined in vitro. The pGI of tortillas decreased, and the values were different depending on the method used to prepare the masa and tortilla. Commercial tortilla and tortilla of NCF had the lowest pGI. Therefore, the procedure to obtain masa and thereafter obtain tortillas influenced the starch digestibility of the product.     

用DNA-RNA杂交检测新城疫病毒的研究

用光敏生物素标记鸡新城疫病毒ｃＤＮＡ，制备核酸探针，经斑点杂交和碱性磷酸酶显色后，探针同该ｃＤＮＡ的ＰＣＲ产物、ＰＣＲ产物重组子和新城疫强弱毒株呈现阳性反应，与ＩＢＶ、ＩＬＴＶ、ＭＧ和正常尿囊液等均呈阴性杂交反应，田间病料的杂交试验也表明，该ｃＤＮＡ探针是敏感且特异的检测方法     

新城疫病毒融合蛋白基因片段的构建与鉴定

本文重组和构建了新城疫病毒 ( NDV)融合蛋白基因 ,并对该基因进行了鉴定 :将新城疫病毒 F基因片段经 RT— PCR扩增 ,插入经 Eco R I/ Sal 酶切的克隆载体 p UC18及表达载体 p GEMEX,转化大肠杆菌 JM10 9株。用氨苄青霉素平板法初步筛选克隆 ,再用双酶切法、核酸探针、 PCR及核苷酸序列分析法鉴定 ,表明插入成功并且阅读框架正确