马立克病毒UL36~（USP）蛋白的表达及其抗体制备

UL36USP是马立克病毒UL36基因编码的蛋白UL36上具有去泛素化酶活性的N-端部分片段（UL36USP）,本试验以提取MDCC-MSB1细胞的基因组为模板通过PCR获得目的基因,将UL36USP基因克隆入pMD18T载体,测序正确并酶切鉴定后,将目的基因亚克隆到pET28a原核表达载体,构建重组质粒pET28a-UL36USP。将重组质粒转化BL21（DE3）E.coli中,IPTG诱导表达并使用Ni-NTA Agarose亲和层析进行纯化得到目的蛋白。应用纯化后的UL36USP免疫獭兔制备多克隆抗体。使用昆虫细胞表达系统表达的UL36片段检测多克隆抗体的特异性。结果显示成功制备特异性的UL36USP抗体。     

马立克氏病毒被膜蛋白VP16(UL48)的表达及多克隆抗体的制备

UL48蛋白是MDV血清Ⅰ型(MDV-Ⅰ)复制所必需,其与MDV编码的另外一种具有去泛素化酶活性的被膜蛋白UL36以及其他被膜蛋白相互作用,为探究MDV编码的UL48与UL36互作在马立克氏病(MD)肿瘤发生机制中作用,本试验制备了致病型强毒MDV-Ⅰ编码的UL48的抗体。从MDV-Ⅰ基因组中克隆了UL48基因,并将测序正确的UL48基因分别亚克隆到pTYB1和pGEX-4T3原核表达载体,构建成pTYB1-UL48和pGEX4T3-UL48重组质粒。将两重组质粒分别转化到BL21(DE3)E.coli中,分别进行IPTG诱导表达,其中pTYB1-UL48表达的融合蛋白Intein-UL48应用Chitin-Sepharose进行亲和层析纯化,将纯化后的融合蛋白Intein-UL48作为免疫大白兔制备多克隆抗体,其中Intein标签有利于提高UL48蛋白的可溶性并提高抗体效价,pGEX4T3-UL48表达纯化的融合蛋白GST-UL48应用凝血酶进行切割得到UL48蛋白,以此为包被抗原,用于间接ELISA和Western blot抗体效价检测和特异性鉴定。结果显示该抗体效价为1:512 000,成功制备特异性的UL48抗体。     

马立克病毒UL36~(USP)蛋白的表达及其抗体制备

UL36USP是马立克病毒UL36基因编码的蛋白UL36上具有去泛素化酶活性的N-端部分片段(UL36USP),本试验以提取MDCC-MSB1细胞的基因组为模板通过PCR获得目的基因,将UL36USP基因克隆入pMD18T载体,测序正确并酶切鉴定后,将目的基因亚克隆到pET28a原核表达载体,构建重组质粒pET28a-UL36USP。将重组质粒转化BL21(DE3)E.coli中,IPTG诱导表达并使用Ni-NTA Agarose亲和层析进行纯化得到目的蛋白。应用纯化后的UL36USP免疫獭兔制备多克隆抗体。使用昆虫细胞表达系统表达的UL36片段检测多克隆抗体的特异性。结果显示成功制备特异性的UL36USP抗体。     

与自噬相关蛋白Becline 1相互作用的猪伪狂犬病毒蛋白的筛选与鉴定

为了对与自噬相关蛋白Becline 1相互作用的猪伪狂犬病毒蛋白进行筛选和鉴定,试验进行了pull-down、Shotgun质谱分析和免疫共沉淀(Co-IP)以及激光共聚焦检测。结果表明:病毒蛋白UL36与Becline 1之间可能存在相互作用关系;UL36-1与Becline 1蛋白之间具有稳定的相互作用关系,相互作用区域位于UL36的去泛素酶区域;UL36-1与Becline 1共定位于胞浆中。说明自噬相关蛋白Becline 1与猪伪狂犬病毒蛋白UL36的去泛素酶区域存在相互作用。     

鸡SUMO1的HIS/GST双融合纯化标签载体的构建及表达分析

SUMO(small ubiquitin-related modifier,小泛素样相关修饰物)是一种重要的蛋白翻译后修饰物,对调节蛋白质的活性、功能和细胞定位都起着至关重要的作用。虽然SUMO和泛素的空间结构很相似,但是发挥的功能却有很大的不同,SUMO不像泛素那样介导蛋白质的降解,反而可以增强蛋白质的稳定性。近年来,研究发现人的SUMO作为一种融合标签和分子伴侣来可以显著增加一些难溶解蛋白的可溶性表达。本研究以原核表达载体pET-28a为基础构建了鸡SUMO1重组质粒HIS-SUMO1-GST-pET28a,并在SUMO1和GST之间插入IL2得到重组质粒HIS-SUMO1-IL2-GST-pET28a,经IPTG诱导表达,并分别使用Ni-NTA及Glutathione-sepharose 2次亲和层析纯化,获得了纯度较高的融合蛋白。活性分析发现去SUMO化酶人的SENP1可完全切开纯化融合的蛋白经。结果表明,此SUMO融合的HIS/GST双标签表达载体不但可以用于提高难溶蛋白的溶解度,而且表达产物也作为去SUMO化酶研究的底物。     

大骨鸡HSP70蛋白的表达、纯化及质量检测

旨在构建鸡热休克蛋白70(heat shock protein 70,HSP70)表达质粒p ColdⅡ-HSP70,诱导表达、纯化后检测HSP70蛋白的质量。将目的基因扩增后克隆至表达载体p Cold II,构建重组载体p ColdⅡ-HSP70,在2种大肠杆菌菌株(BL21(DE3)和Rosetta(DE3)p Lys S中经IPTG在不同温度与不同浓度条件下诱导表达并纯化。经SEC-FPLC和HPLC进行纯度鉴定,采用鲎试剂检测蛋白内毒素含量(将HSP70蛋白按1 200、600、120倍稀释);Western blot及质谱方法检测蛋白分子量。结果显示:酶切和测序结果均证实HSP70表达载体构建正确,可诱导表达。经IPTG诱导表达后确定BL21(DE3)在37℃条件下表达蛋白效果最佳,经检测HSP70蛋白的相对分子质量为71 988.04,蛋白纯度大于90%,内毒素小于500 Eu/mg。结果表明:成功构建了大骨鸡HSP70基因的表达载体,获得了纯化的HSP70蛋白,为进一步研究HSP70在热应激中的作用机制提供了基础。     

鸡chUSP1 His594Ala突变使其去泛素化酶活性丧失

泛素特异性蛋白酶1(ubiquitin-specific proteases 1,USP1)是一种去泛素化酶,它在DNA损伤修复应答过程中发挥重要功能。已证明USP1与UAF1(USP1-associated factor 1)形成复合物后,其酶活性会得到极大的提升。chUSP1为在鸡细胞中发现的一种去泛素化酶,生物信息学分析发现它与人USP1同源性高达72%。本研究以本室扩增的chUSP1基因和chUAF1基因,使用bac-to-bac系统表达了chUSP1GG680,681AA单体及chUSP1GG680,681AA/chUAF1复合体。并根据人USP1预测将chUSP1一个可能的活性中心位点第594位组氨酸突变为丙氨酸,并纯化了此突变型的单体和复合体。应用底物Di-Ub/Ub2 WT Chains(K63-linked)及Di-Ub/Ub2 WT Chains(K48-linked)对chUSP1GG680,681AA和突变型chUSP1HGG594,680,681AA进行酶活性分析。试验结果表明chUSP1具有切割Ub链底物的酶活性,且第594位组氨酸残基突变影响chUSP1的去泛素化酶活性。而chUAF1可以与chUSP1相互作用形成复合体,并且明显提高chUSP1酶活性。     

鸡β-防御素-2(Gal-2)基因在大肠杆菌中的融合表达与纯化

本试验通过优化基因工程菌pMAL-c2X-Gal-2-TB1的表达条件,找到使融合蛋白表达量达到最大的最佳表达条件,后采用Amylose树脂预装柱对可溶性目的蛋白分离、纯化和Fac-tor Xa因子酶切,为了得到Gal-2,再次过柱纯化,每一步产物用SDS-PAGE方法检测.结果融合蛋白的分子量约为50KD,融合蛋白的表达量约占全菌总蛋白的36%;纯化后的融合蛋白纯度达90%以上,且经再次纯化得到纯度较高的Gal-2.     

禽白血病病毒p27蛋白在大肠杆菌中的表达和多克隆抗体的制备

将禽白血病病毒p27基因克隆并构建重组表达质粒pET28a-p27,在大肠杆菌BL21中经IP TG诱导后产生可溶性表达蛋白。表达的蛋白用Western blot进行活性检测,其能与辣根过氧化物酶标记的兔抗p27抗体发生特异性反应;采用金属螯和层析对表达蛋白进行纯化,其纯度约为95％;用纯化的表达蛋白p27免疫家兔制备抗血清,ELISA抗体效价可达1∶25600。研究结果表明,大肠杆菌表达的p27蛋白具有良好的抗原性,可以替代纯化病毒蛋白用于禽白血病病毒检测。     

重组牛α-干扰素的原核表达与纯化

为在大肠杆菌表达系统中高效表达牛α-干扰素(bovine interferon-α,Bo IFN-α),将经密码子优化后的牛α-干扰素成熟蛋白基因合成并克隆到表达载体p Cold II中,转化大肠杆菌BL21(DE3)中诱导表达,获得可溶性的表达蛋白。对重组菌表达条件进行优化,结果显示在IPTG浓度为0.64 mmol/L,诱导温度为15℃,诱导时间为9 h的条件下,可溶性目的蛋白的表达量最高。重组蛋白经镍柱纯化后的纯度为99.2%,表达量为36 mg/L菌液。重组蛋白在MDBK细胞上抗水泡性口炎病毒的活性为1×106U/mg。     

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。     

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.     

Mechanism of Action of Probiotic Function of Lactobacilli

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。 …