金黄色葡萄球菌FnBA活性基因的克隆及其原核表达

根据GenBank中纤连蛋白结合蛋白A基因(FnBA)序列设计了1对特异性引物,以金黄色葡萄球菌基因组DNA为模板,进行PCR扩增;结果获得了1 735 bp的DNA片段.将PCR产物克隆至pMD18-T载体中,成功地构建了克隆质粒pMD18-T-FnBA.以HindⅢ和BamH Ⅰ双酶切pMD18-T-FnBA和pET28a(+),将纯化的基因FnBA亚克隆至pET28a(+)中,构建了原核表达质粒pET28a-FnBA,并将其转化至E.coli BL21感受态细胞中,经1 mmol/L IPTG诱导和SDS-PAGE分析,在约85 000处出现了与预期目的蛋白一致的外源蛋白带.又经Western-blotting分析表明,该蛋白具有金黄色葡萄球菌的抗原性.     

奶牛乳房炎金葡菌黏附素亚单位疫苗的基础研究

为构建金黄色葡萄球菌黏附素基因原核表达质粒,采用PCR方法对凝集因子A（ClfA）的A区、纤连蛋白（FnBPs）A和B的D区基因进行特异性扩增,构建了克隆质粒pMD19T-ClfA、pMD19T-FnBPA和pMD19T-FnBPB,然后将克隆基因定向插入到原核表达载体pET32a（＋）中,构建了表达质粒pET32-ClfA、pET32-FnBPA和pET32-FnBPB,并将其转入宿主菌BL21 （DE3）,经SDS-PAGE分析表明,1 mmol/L IPTG诱导后,在57ku、35ku和35ku处出现了与目的蛋白一致的外源蛋白带;经western-blot分析表明,所表达的蛋白均与目的蛋白一致,说明外源基因成功表达,且表达产物具有良好的免疫原性.     

金黄色葡萄球菌纤连蛋白结合蛋白A基因的克隆及其原核表达

根据GenBank中纤连蛋白结合蛋白A基因（fnbA）序列设计了1对特异性引物，以金黄色葡萄球茵基因组DNA为模板，进行PCR扩增；结果获得了3600bp的DNA片段。将PCR产物克隆至pGEM T easy载体中，成功地构建了克隆质粒pGEM—fnbA。以HindⅢ和XhoⅠ双酶切pGEM-fnbA和pET28a（＋），将纯化的基因fnbA亚克隆至pET28a（＋）中，构建了原核表达质粒pET28a-fnbA，并将其转化至E．coli BL21（DE3）感受态细胞中，经1mmol／LIPTG诱导和SDSPAGE分析，在约165ku处出现了与预期目的蛋白一致的外源蛋白带。Western—blotting分析表明，该蛋白具有金黄色葡萄球菌的抗原性。     

牛分枝杆菌mpb51-63融合基因的克隆及其原核表达

为提高牛分枝杆菌(M.bovis)单一抗原的抗原性,本研究以重叠延伸拼接PCR技术将M.bovis mpb51与mpb63基因连接,并克隆至pMD18-T中,构建重组质粒pMD-51-63。以BamHⅠ和EcoRⅠ双酶切pMD-51-63和pET28a(+),将纯化的mpb51-63融合基因亚克隆至pET28a(+)中,获得了重组表达质粒pET-51-63。该重组质粒在大肠杆菌BL21(DE3)中经IPTG诱导和SDS-PAGE分析,表达约46 ku的外源融合蛋白,免疫印迹实验证实,该融合蛋白具有M.bovis的抗原反应性。为进一步研究mpb51-63融合基因及其表达产物作为牛结核病亚单位疫苗、核酸疫苗以及特异的诊断试剂奠定了基础。     

奶牛乳腺炎源性金黄色葡萄球菌山东分离株β-溶血素基因的克隆及原核表达

以提取的奶牛乳腺炎源性金黄色葡萄球菌山东分离株(zfb)全基因组DNA为模板,PCR扩增β-溶血素基因(hlb),并与克隆栽体pMD18-T相连接.测序结果表明,扩增片段含有993 bp的ORF,可编码含330个氨基酸的成熟蛋白,与已报道的金黄色葡萄球菌β-溶血素蛋白的氨基酸同源性为99.4%.构建原核表达载体pET32a+/hlb,SDS-PAGE分析蛋白表达水平,IPTG诱导后表达的融合蛋白的相对分子量质为57 000,表达量占菌体总蛋白的23.9%;表明原核表达质粒构建成功,并实现了有效表达.     

牛分枝杆菌MPB70基因的克隆及其在大肠杆菌中的表达

本研究以牛分枝杆菌Vallee111染色体DNA为模板,以MPB70成熟蛋白基因特异性引物进行PCR扩增,获得约500bp的DNA片段.通过T-A克隆技术,将PCR产物克隆至pGEM-T Vector中,成功地构建出克隆载体pGEM-T-70.以BamH Ⅰ和EcoR Ⅰ双酶切pGEM-T-70和pET28a( ),并将纯化的MPB70基因亚克隆至pET28a( )中,构建出原核表达载体pET28a-70.将pET28a-70转化至感受态E.coli BL21(DE3)中,经IPTG诱导和SDS-PAGE分析,可见约25Ku外源蛋白带.Western blot分析发现,该蛋白具有牛分枝杆菌抗原性,从而为进一步研究MPB70的亚单位疫苗及DNA疫苗奠定基础.     

犬瘟热病毒贵州株融合蛋白基因原核表达质粒的构建

为了研究犬瘟热病毒贵州株(CDV-GZ1)完整融合蛋白(F),试验采用PCR方法以pMD18-F质粒为模板,利用特异性引物扩增获得大小为1 989 bp的目的 DNA,并将其克隆至pET32a(+)原核表达载体中,获得重组质粒pET32a(+)-F。结果表明:目的基因插入位置和阅读框均正确,说明F基因原核表达质粒构建成功;质粒pET32a(+)-F在BL21(DE3)中经诱导表达未获目的蛋白,说明CDV融合蛋白可能不适合在该表达系统中进行完整蛋白的表达。     

胸膜肺炎放线杆菌APXⅣ部分基因片段的表达及纯化

参照GenBank中已发表的ApxⅣ基因序列,以自行分离的App DNA为模板,利用PCR方法扩增出ApxⅣ3′端,大小为552bp的保守基因序列。将PCR产物克隆到pMD18-T Simple Vector中,获得重组质粒pMD-ApxⅣ,对其重组质粒pMD—ApxⅣ进行BamHⅠ、HindⅢ双酶切,并将酶切产物克隆到原核表达载体pET-32a（＋）中,构建了重组表达质粒pET—ApxⅣ。将表达质粒转化至大肠杆菌BL21中,用IPTG诱导表达,通过SDS-PAGE和Western blot分析,结果表明pET—ApxⅣ在BL21中成功表达,并能被App阳性血清所识别,具有良好的免疫原性。表达蛋白的分子质量约为39．5KOa。利用HiTrap FF crude columns将表达的蛋白进行了纯化。     

奶牛乳腺炎金黄色葡萄球菌ClfA核酸疫苗的构建及免疫原性试验

扩增了奶牛乳腺炎金黄色葡萄球菌ClfA基因,并将其克隆至真核表达载体pVAX1启动子下游,构建成真核表达质粒,通过体外细胞转染试验,运用IFA方法进行抗原性初步确认,所构建的重组DNA疫苗质粒能在真核细胞中表达并被金黄色葡萄球菌抗体特异性识别。为进一步评价侯选疫苗的免疫原性,进行了BALB/c小鼠免疫试验,分别检测免疫后的ELISA抗体水平、Th1/Th2类细胞因子水平以及T淋巴细胞增殖试验。结果表明,构建的核酸疫苗pVAX1-ClfA免疫小鼠后,ELISA抗体水平提高,Th1/Th2类细胞因子含量提升,T细胞增殖能力增强。     

牛分枝杆菌Ag85B基因的克隆及其在大肠杆菌中的表达

以牛分枝杆菌Vallee111染色体DNA为模板．根据已发表的Ag85B成熟蛋白基因的核苷酸序列设计合成特异性引物进行PCR扩增．获得约860bp的DNA片段。通过T-A克隆技术．将PCR产物克隆至pGEM-T载体中．成功地构建出克隆载体pGEM-T-85B。以BamHⅠ和EcoRⅠ双酶切pGEM-T-85B和pET28a（＋）．并将纯化的Ag85B基因亚克隆至pET28a（＋）中，构建出原核表达载体pET28a-85B。将pET28a-85B转化至感受态E.coli BL21（DE3）中．经IPTG诱导和SDS-PAGE分析．可见约30000的外源蛋白带。Western blot分析发现，该蛋白具有牛分枝杆菌抗原性．从而为进一步研究Ag85B的亚单位疫苗及DNA疫苗奠定了基础。     

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.