抗冻基因CBF_2表达载体构建及转化紫花苜蓿的研究

本研究以拟南芥基因组DNA为模板,用PCR方法扩增目的基因,连接到PGEM-T Easy Vector载体上构建成克隆载体T-CBF2。用BamHⅠ和SacⅠ分别对克隆载体T-CBF2和植物表达载体PBI121进行双酶切,获得目的片段和线性质粒。在T4DNA连接酶的作用下进行定向连接,构建成植物表达载体P-T-CBF2。采用直接转化法将重组子导入根癌农杆菌EHA105。经PCR鉴定,重组质粒已成功导入根癌农杆菌中。通过农杆菌介导法转化和田苜蓿,现在已经得到转基因的苜蓿愈伤组织。     

利用BrdU在CEF（TK^＋）细胞中筛选TK^—重组鸡痘病毒

将新城疫病毒（NDV）四平株HN基因，插入不含报告基因，以TK为侧翼的鸡痘病毒表达载体pUTA-2中的复合启动子下游，获得重组表达质粒pUTA-2HN。将重组表达质粒转染鸡痘病毒（FPV）感染的鸡胚成纤维细胞（CEF），培养、收获病毒后，用含40mg/L5－溴－2－脱氧尿嘧啶（BrdU)的培养液，在CEF（TK^ )细胞中筛选培养2代，然后用不含BrdU的培养液进行病毒蚀斑纯,化成功筛选出表达NDV HN蛋白的TK^-重组鸡痘病毒。     

TRAIL和HN基因融合表达重组腺病毒的构建及其对DT40细胞的抑瘤作用

分析携TRAIL和HN基因的重组腺病毒在DT40细胞中的表达规律及杀伤肿瘤细胞的生物学效应。通过RT-PCR分别从鸡外周血淋巴细胞和新城疫病毒(NDV)LaSota株中扩增出TRAIL和HN基因,通过定点突变和重叠延伸拼接法实现TRAIL和HN基因的2A连接,将获得的目的基因克隆到pShuttle-CMV穿梭载体中,与pAdeasy-1载体在BJ5183细菌内同源重组,构建了包含目的基因的重组腺病毒质粒pAd-TRAIL和pAd-TRAIL-2A-HN,将质粒线性化后转染AD293细胞,包装后的重组腺病毒,经RT-PCR检测、荧光显微镜观察和病毒滴度测定后,将获得的重组腺病毒体外转染禽淋巴白血病DT40细胞,通过RT-PCR和Western blotting检测重组腺病毒的感染性和外源基因的表达情况,采用流式细胞仪检测重组腺病毒对DT40细胞生长的影响以及Ad-TRAIL和Ad-TRAIL-2A-HN对DT40细胞的凋亡作用。PacⅠ酶切证实同源重组成功;RT-PCR、荧光显微镜检测证实重组腺病毒质粒成功导入AD293细胞中且具有良好的感染性,滴度为1.9×1010PFU/mL;Western blot检测结果显示,TRAIL-linker-HN基因能够在DT40细胞中稳定表达并对DT40细胞产生细胞毒作用,且诱导细胞的凋亡率为29.52%,表明TRAIL-linker-HN具有双基因抑瘤的协同效应。     

利用BrdU在CEF(TK~ )细胞中筛选TK~-重组鸡痘病毒

将新城疫病毒 (NDV)四平株 HN基因 ,插入不含报告基因、以 TK为侧翼的鸡痘病毒表达载体 p UTA- 2中的复合启动子下游 ,获得重组表达质粒 p UTA- 2 HN。将重组表达质粒转染鸡痘病毒 (FPV)感染的鸡胚成纤维细胞 (CEF) ,培养、收获病毒后 ,用含 40 m g/ L 5 -溴 - 2 -脱氧尿嘧啶 (Brd U )的培养液 ,在 CEF(TK )细胞中筛选培养 2代 ,然后用不含 Brd U的培养液进行病毒蚀斑纯化 ,成功筛选出表达 NDV HN蛋白的 TK-重组鸡痘病毒     

表达鸡传染性法氏囊病病毒变异株VP2基因的重组嵌合型新城疫病毒的构建与鉴定

为了研发防控IBDV变异株和基因Ⅶ型NDV感染的高效廉价多联疫苗,将IBDV中国流行变异株的VP2基因共有序列插入到含有基因Ⅶ型NDV毒株F和HN基因的嵌合型NDV LaSota-ⅦF/HN全长基因组cDNA的质粒pNDFL-ⅦF/HN中,构建含有IBDV变异毒株VP2基因的重组NDV全长感染性克隆pNDFL-ⅦF/HN-VP2。将pNDFL-ⅦF/HN-VP2和辅助质粒共转染BHK-21细胞拯救重组NDV rChiLaSota-VP2。利用PCR和Western blot对重组NDV进行鉴定,并对重组病毒的生物学特性进行研究。结果显示,重组NDV rChiLaSota-VP2拯救成功并可以正确表达IBDV VP2蛋白。第25代重组NDV在鸡胚中的生物学特性研究表明,rChiLaSota-VP2保留了LaSota株在鸡胚中高滴度复制、低致病性和遗传稳定性等生物学特性。重组NDV rChiLaSota-VP2的鸡胚半数感染量(EID₅₀)峰值可达10^-8.16/100μL,鸡胚平均致死时间(MDT)为134 h, 1日龄雏鸡脑内接种致病指数(I...     

以减毒鼠伤寒沙门氏菌为载体的重组NDV-HN口服疫苗的构建与鉴定

试验旨在构建表达鸡新城疫病毒(NDV)HN基因的重组减毒鼠伤寒沙门氏菌SL1344ΔcrpΔasd(pYA3493-HN)。以pMD18-T-HN为模板,通过PCR扩增出NDV HN基因片段,定向插入原核表达载体pYA3493中,将重组表达质粒pYA3493-HN转入χ6097,再转入减毒鼠伤寒沙门氏菌SL1344ΔcrpΔasd,通过双酶切和PCR对质粒进行鉴定。结果表明,携带NDV HN基因片段的重组减毒鼠伤寒沙门氏菌SL1344ΔcrpΔasd(pYA3493-HN)构建成功。本研究结果为开发鸡新城疫的口服基因工程活载体疫苗奠定了基础。     

东方山羊豆GoGID基因表达载体构建及紫花苜蓿转化

根据已经克隆得到的东方山羊豆赤霉素受体(GoGID)基因,扩增编码区cDNA.以pBI121为基础载体,采用酶切连接法,构建植物超表达载体pBI121-GoGID.酶切鉴定表明:目的基因已经正确插入载体中,超表达载体构建成功.采用CaCl₂冻融法将重组载体转入农杆菌菌株中.以叶片为外植体,采用农杆菌介导的愈伤组织培养法,转化紫花苜蓿(Medicage sativa),得到抗性苗.对载体携带的nptⅡ基因、GUS基因进行PCR检测均成阳性,表明目的基因已成功导入紫花苜蓿基因组中.同时对转基因植株进行Southern-blot及RT-PCR检测,并均得到目的条带.本研究为进一步分析GoGID基因对紫花苜蓿生物量影响奠定了基础.     

aFGF植物表达载体构建及转化紫花苜蓿的初步研究

主要将aFGF基因按照植物偏爱的密码子进行基因优化,同时加上信号肽、6-组氨酸标签及凝血酶切割因子,合成全基因,再将基因插入到改造的TΩ4AB质粒中,将aFGF基因和质粒上的增强子、启动子、Ω序列及ployA加尾序列双酶切,命名为TΩ4AB-aF,将TΩ4AB-aF插入到pBI121载体中命名为pBI121-TΩ4AB-aF。利用三亲融合法将pBI121-TΩ4AB-aF转入到农杆菌菌株LBA4404中,转化苜蓿。转基因苗用卡那霉素进行筛选;并对抗性苗进行PCR、RT-PCR、Western Blot检测。结果表明,aFGF在苜蓿中得到表达。为苜蓿作为植物生物反应器奠定理论基础。     

基因Ⅱ、Ⅶ型二价新城疫病毒样颗粒的制备和鉴定

将基因Ⅶ型新城疫NA-1株F、HN基因的胞外域与基因Ⅱ型新城疫Lasota株F、HN基因的胞内域及跨膜域进行融合,并命名为rNA-1 F/HN。利用昆虫杆状病毒表达系统构建含有新城疫LaSota株M、F、HN等3个结构基因及rNA-1F、rNA-1 HN基因的重组杆状病毒rBV-LaSota M、rBV-LaSota F、rBV-rNA-1 F、rBV-LaSota HN、rBV-rNA-1 HN。将几种重组杆状病毒共感染Sf9细胞96h后收取上清,经超速离心及蔗糖密度离心纯化病毒样粒子(virus-like particles,VLPs),并利用Western blot方法和透射电子显微镜技术检测,结果显示目的蛋白均正确表达且组装成与野生型NDV形态大小相似的VLPs。结果表明:成功制备了新城疫标准疫苗株(LaSota株)及流行强毒株(NA-1株)二价NDV VLPs,为当前新城疫的防控提供新策略。     

牛β-干扰素在新城疫病毒La Sota疫苗株中的高效稳定表达及抗病毒活性检测

研究利用新城疫病毒La Sota株作为表达载体,构建表达牛β-干扰素基因的重组新城疫病毒全基因组质粒pFL-BoIFN-β, 与辅助质粒共转染BHK-21细胞后获得表达BoIFN-β的重组新城疫病毒.收获的鸡胚尿囊液内的重组病毒经RT-PCR检验证实重组病毒含有相应外源基因.将重组病毒尿囊液经紫外线照射以灭活新城疫病毒,利用表达绿色荧光蛋白的重组水泡性口炎病毒在牛肾细胞上测定rL-BoIFNβ抗病毒活性, 表达BoIFN-β的重组新城疫病毒能有效抑制重组水泡性口炎病毒在牛肾细胞上的复制, rL-BoIFNβ接种SPF鸡胚所收尿囊液抗病毒活性高达2×107 IU/mL.结果表明:重组新城疫病毒rL-BoIFNβ接种的SPF鸡胚尿囊液具有高效而稳定的抗病毒活性,重组新城疫病毒可以作为外源蛋白的高效表达载体而广泛应用.     

商会自治的基石——商会自治规范研究

在现代法律秩序中,商会自治规范是制定法的基础和必要的补充,甚至在某些方面替代了制定法;商会自治规范主要包括商会组织规范、行为规范、惩罚规范以及争端解决规范等;其效力仅及于其内部成员;商会自治规范和制定法之间存在冲突,但也存在整合的基础。     

癸氧喹酯干混悬剂含量测定的改进

采用高效液相色谱法测定癸氧喹酯干混悬剂的含量,在2-250μg/mL范围内,峰面积的常用对数与进样量浓度的常用对数呈良好的线性关系,R^2=1(n=5),平均回收率为99.24%~99.51%,RSD在0.05%~0.28%。此方法分析时间短,样品前处理简便、定量结果准确,重现性好,结果满意,为其质量控制提供了依据。     

猪毛色遗传的研究进展

本文概述了猪的毛色类型、猪的毛色遗传模式,着重综述了猪毛色基因分子基础的研究进展,指出存在问题并就未来发展方向做了思考。     

Comparison of 2 methods of centesis of the bursa of the biceps brachii tendon of horses

REASONS FOR PERFORMING STUDY: Centesis of the bicipital bursa using an 8.9 cm long spinal needle has been reported but the alternative of employing a 3.8 cm long hypodermic needle requires validation. OBJECTIVE: To compare the efficacy of 2 different methods of centesis of the bicipital bursa and to evaluate the usefulness of ultrasonographic imaging to determine the location of solution administered when centesis of the bursa is attempted. METHODS: For Trial 1, 6 clinicians, who had no previous experience of centesis of the bicipital bursa, attempted to inject a solution composed of an aqueous radiopaque contrast medium and physiological saline solution (PSS) into the bicipital bursae of 2/12 horses using the previously described distal approach to inject one bursa and a proximal approach to inject the contralateral bursa. The bicipital tendon and bursa were examined ultrasonographically before and after injection; and both shoulders were examined radiographically to identify the location of the medium. In Trial 2, another 6 clinicians, also with no previous experience of centesis, repeated Trial 1, using 6 horses, but the radiopaque contrast medium was mixed with air instead of PSS. RESULTS: Accuracy of centesis using the proximal approach was 39% and that of the distal approach 28%. Ultrasonographic examination of the shoulder allowed the location of solution and air to be accurately predicted in all 12 shoulders examined. CONCLUSIONS: Clinicians who have had no previous experience performing centesis of the bicipital bursa are unlikely to be successful in centesis using either approach. Radiographic examination after injecting a radiopaque contrast medium may be necessary to assess the success of centesis especially if bursal fluid is not obtained during centesis. Injecting air along with the radiopaque contrast medium provides more accurate ultrasonographic confirmation of centesis and better radiographic definition than does injection without air.     

不同样本商品蜂蜜中硒含量的测定

用硝酸和高氯酸消化蜂蜜,使硒游离出来,在微酸性环境下,硒和2,3-二氨基萘(DAN)生成有较强荧光的物质,用环己烷萃取,在激发波长378nm,荧光波长518nm处测定其荧光强度。蜂蜜中硒含量范围:0.10～0.82μg/g。表明:蜂蜜应视为天然富硒营养品。     

乳酸杆菌益生作用机制的研究进展

乳酸杆菌作为益生菌广泛用于人和动物。本文综述了乳酸杆菌改善宿主健康的机制。乳酸杆菌可通过产生抗菌物质如乳酸、过氧化氢、细菌素,或者通过竞争营养或肠道黏附位点来抑制致病菌;通过诱导黏附素的分泌或阻止细胞凋亡而增强肠道的屏障功能,从而保护肠道。文章重点讨论了乳酸杆菌表面成分（表面蛋白、脂磷壁酸和肽聚糖）与肠道受体（Ｃ型凝集素受体、Ｔｏｌｌ样受体和 Ｎｏｄ样受体）,阐述了他们结合后启动免疫调节信号,调控肠道免疫功能以发挥改善健康作用的机制。     

中华人民共和国农业部公告 第267号

为贯彻落实《兽药生产质量管理规范》(简称《兽药GMP》),进一步推动兽药GMP实施进程,我部制定了《兽药生产质量管理规范检查验收办法》,现予公告。本公告自2003年6月1日起施行。附件:兽药生产质量管理规范检查验收办法二○○三年四月十日第一章 总则 第一条 为推动《兽药生产质量管理规范》(以下简称兽药GMP)的实施,规范兽药GMP检查验收工作,制定本办法。 第二条 农业部负责全国兽药GMP管理和检查验收工作;负责制修订兽药GMP检查验收管理规定;负责兽药GMP检查员队伍建设和监督管理工作,负责国际兽药贸易中GMP互认工作。 …     

鸡败血支原体垂直传播模式及其阻断方法

以国际标准强毒Ｒ株人工感染非免疫产蛋鸡,定时扑杀,分别从鼻窦、眶下孔、气管、肺、气囊、卵巢和输卵管分离ＭＧ,并收集感染鸡所产蛋分离ＭＧ。结果表明,人工感染４８小时后上、下呼吸道及肺已被全面感染,９６小时气囊已被感染,１２０小时输卵管已能分离到ＭＧ,卵巢始终分离不到ＭＧ。人工感染鸡自１４４小时便能在其所产蛋中分离出ＭＧ。药物治疗能在７２小时内消除感染,油乳剂苗则需２４天后逐渐降低蛋内ＭＧ分离率,药物卵内注射、种蛋药浴、高温处理均能杀死卵内ＭＧ,但以研制的种蛋浸泡剂药浴效果为最好。     

Effects of size of ingestively masticated fragments of plant tissues on kinetics of digestion of NDF

Ingestively masticated fragments were collected and sized via sieving. Different sizes of esophageal masticate and ruminal digesta fragments, and ground fragments of larger masticated pieces were incubated in vitro, and undigested NDF remaining at intervals of up to 168 h of incubation was determined. The ruminal age-dependent time delay (tau) for onset of digestion of NDF was positively correlated (P < 0.004) with the mean sieve aperture estimated to retain 50% of the fragments between successive sieve apertures (MRA). Degradation rate of potentially degradable NDF (PDF) and level of indigestible NDF were not related (P > 0.10) to MRA of masticated and ground fragments. Estimates of tau were positively related to MRA, with slopes of bermudagrass < corn silage < ruminal fragments of corn silage. It was concluded that fragment size-, and consequently, ruminal age-dependent onset of PDF degradation of a mixture of different fragment sizes results in an age-dependent rate of degradation of the more rapidly degrading of two subentities of PDF. Models are proposed that assume a tau before onset of simultaneous degradation of PDF from two pools characterized as having gamma-modeled age-dependency and age-constant rates. The ruminal age-dependent pool seems to be associated with the faster-degrading pool, and its rate parameter increases with range in MRA in the population of fragments. Conceptually, the ruminal age-dependent rate parameter for PDF degradation seems to represent a composite of several effects: 1) effects of the size-dependent tau; 2) range in MRA of the population of ingestively masticated fragments; and 3) subentities of PDF that degrade via more rapid age-dependent rates compared with subentities of PDF that degrade via age-constant rates. The estimated fractional rates of ruminative comminution of ingestively masticated fragments (0.060 to 0.075/h) were of a magnitude similar to the mean fractional rates of PDF digestion (0.030 to 0.085/h), which implies that ruminative comminution may be first-limiting to fractional rate of PDF digestion. The in vivo roles of ingestive and ruminative mastication of fragments on PDF degradation must be considered in any kinetic system for estimating PDF digestion in the rumen. These results and others in the literature suggest that the rate of surface area exposure rather than intrinsic chemical attributes of PDF may be first-limiting to degradation rate of PDF in vivo.