大肠杆菌多药耐药基因AcrA的克隆及其原核表达

从大肠杆菌AcrA的编码序列中设计引物，以大肠杆菌基因组为模板，扩增出AcrA基因中约691bp的cDNA片段，将所得片段与pMD—18T载体连接，转化到DH5α大肠杆菌中，成功地筛选到阳性克隆，其质粒测序结果与文献报道一致。从阳性克隆中提取质粒，经HindⅢ和BamHI酶解，回收691bp的目的片段，定向克隆到pET—28a表达载体中，提取质粒，再次转化到BL21(DE3)中，成功地筛选出阳性克隆。经IPTG诱导阳性菌，通过SDS—PAGE检测出AcrA部分基因的表达。     

大肠杆菌acrA基因的克隆、表达、抗体制备及不同耐药水平大肠杆菌AcrA表达水平的检测

检测不同耐药水平大肠杆菌AcrA表达水平，探讨耐药水平与AcrA蛋白表达水平之间的关系。对acrA基因进行克隆、表达、制备抗AcrA抗体，Western blot方法比较同耐药水平大肠杆菌AcrA表达水平。结果表明，多重耐药株SEMR的AcrA表达明显高于单药耐药株SEICI和SEICH以及质控株ATCC25922。因此SEMR多重耐药性的产生与AcrA的高效表达直接有关。     

大肠杆菌多药耐药基因AcrA的克隆及其原核表达

从大肠杆菌AcrA的编码序列中设计引物,以大肠杆菌基因组为模板,扩增出AcrA基因中约691 bp的cDNA片段,将所得片段与pMD-18T载体连接,转化到DH5a大肠杆菌中,成功地筛选到阳性克隆,其质粒测序结果与文献报道一致.从阳性克隆中提取质粒,经HindⅢ和BamH I酶解,回收691 bp的目的片段,定向克隆到pET-28a表达载体中,提取质粒,再次转化到BL21(DE3)中,成功地筛选出阳性克隆.经IPTG诱导阳性菌,通过SDS-PAGE检测出AcrA部分基因的表达.     

猪圆环病毒2型H株cap基因原核表达载体的构建与表达

利用PCR技术从临床病料中提取病毒DNA,扩增出猪圆环病毒PCV-2核衣壳蛋白(Cap)基因。将其克隆到原核表达载体pET-32a中,构建了表达载体pET-H2,转化至大肠杆菌BL21中,用ITPG诱导表达。SDS-PAGE分析显示,重组蛋白主要以不溶性包涵体形式表达,与预期大小30 ku相符;Western-blotting分析表明,表达产物能与抗猪PCV2阳性血清发生反应。     

大肠杆菌多重耐药调控基因evgS的克隆及其原核表达

以大肠杆菌ATCC25922的基因组为模板,根据GenBank中大肠杆菌的evgS基因序列设计引物,PCR扩增出约894 bp的基因片段,将所得片段与pMD18-T simple vector连接,转化至大肠杆菌JM109中,筛选阳性克隆,其质粒中插入序列测序结果与GenBank中报道一致.提取阳性克隆质粒,经EcoR Ⅰ和Xho Ⅰ双酶切,回收目的片段,定向克隆到pET-28a原核表达载体中,提取阳性质粒,转化至大肠杆菌BL21(DE3)中,获得阳性克隆.经IPTG诱导阳性菌,收集表达产物,通过SDS-PAGE分析证实,pET-28a-evgS可成功地在大肠杆菌中表达EvgS蛋白.Western-blot检测证明,表达产物具有良好的反应原性.     

禽白血病病毒衣壳蛋白p27基因的原核表达及多克隆抗体的制备

从J亚群禽白血病病毒(ALV-J)HB09JY03株感染的DF-1细胞的冻融裂解液中提取DNA,通过PCR方法扩增出约720bp的gag-p27基因片段,克隆至pMD18-T载体。鉴定正确后,将该片段克隆至pET-30a(+)原核表达载体,经IPTG诱导表达。表达产物经His Trap TMHP纯化后,测蛋白浓度达到9mg/mL。用兔抗自然p27蛋白阳性血清进行Western blot检测,表明重组p27蛋白有抗原反应活性。免疫新西兰大白兔后,产生的抗体与禽白血病全病毒抗原可以发生特异性反应。所制备的重组p27蛋白和多克隆抗体将在ALV的抗原分析、血清学诊断等方面有着重要的应用价值。     

烟碱酰胺合成酶基因的原核表达及多克隆抗体制备

以重组克隆质粒p0390-MF-NASHOR1为模板,通过聚合酶链式反应(polymerase chain reaction,PCR)扩增出烟碱酰胺合成酶基因(nas)片段并将其克隆到原核表达载体pGEM-KG中,获得重组表达质粒pKG-NAS。将此重组表达载体质粒转化到大肠杆菌DH5α中,经异丙基-β-D硫代半乳糖苷(isopropyl-β-D-thiogalactopyranoside,IPTG)诱导表达。聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-poly acrylamide gel electrophoresis,SDS-PAGE)结果显示,烟碱酰胺合成酶基因在大肠杆菌中成功表达,表达的烟碱酰胺合成酶融合蛋白分子量大约为61 kDa。将初步的纯化产物作为免疫原去免疫新西兰大白兔制备兔抗烟碱酰胺合成酶血清,用蛋白免疫印迹法(western blot-ting)对制备的兔抗血清中的多克隆抗体进行了鉴定,显示该抗体具有较好的特异性。烟碱酰胺合成酶基因多克隆抗体的成功制备为检测该基因在草坪草中的表达提供了可靠的手段和技术平台,为进一步阐明该基因的作用机理奠定了基础。     

鸡BCL10基因的克隆与表达

通过电子克隆手段,克隆到大小为959bp的cDNA序列。然后从鸡的脾脏提取RNA,用RT-PCR方法扩增出目的片段。将该片段连接到原核表达载体pET-32a和真核表达载体pEGFP-C1中,采用SDS-PAGE检测原核表达载体pET-BCL10在大肠杆菌中的表达,脂质体转染法将真核表达载体pEGFP-BCL10转入vero细胞,Western blotting检测其在细胞中的表达。结果显示,PCR扩增到735bp的DNA片段,原核表达载体和真核表达载体经双酶切和核酸测序表明载体构建成功。SDS-PAGE电泳检测到pET-BCL10重组质粒在大肠杆菌中表达。Western blotting检测到pEGFP-BCL10重组质粒在vero细胞中表达。结论表明,BCL10基因在鸡体内存在,该基因的重组质粒能在大肠杆菌和vero细胞中表达。     

马驽巴贝斯虫新疆伊犁株Bc48基因克隆、表达及其多克隆抗体的制备

为表达马驽巴贝斯虫新疆伊犁株Bc48基因,制备多克隆抗体,试验根据GenBank中马驽巴贝斯虫Bc48基因序列,设计合成1对特异性引物,以提取的新疆株Bc48基因组DNA为模板进行PCR扩增,将扩增产物克隆至原核表达载体pET-28a（+）中,阳性质粒转化至大肠杆菌BL21（DE3）感受态细胞中,经IPTG诱导后表达产物进行SDS-PAGE分析,切胶纯化蛋白后免疫6周龄雌性BALB/c小鼠,制备多克隆抗体;同时把该基因克隆至真核表达载体pCMV-N-flag中,将阳性质粒转化至大肠杆菌DH5α感受态细胞中,提取pCMV-N-flag-Bc48质粒转染到293T细胞中进行真核表达。结果显示,获得了大小约为15 ku的融合蛋白,与预期目的蛋白大小相一致;免疫BALB/c小鼠制备的多克隆抗体经ELISA和Western blotting检测、验证,能特异地识别相应抗原,其抗体效价可达1：128 000,说明该多克隆抗体有较高的抗体效价和特异性;真核表达质粒在293T细胞中表达Bc48蛋白。本试验可为进一步研究马驽巴贝斯虫功能基因及建立快速的检测方法奠定基础,在虫株的分类学研究及候选疫苗的研制中有重要意义。     

猪细小病毒VP2的原核表达与多克隆抗体制备

克隆猪细小病毒(PPV)VP2基因全长1 740bp,构建其原核表达载体后进行蛋白的表达与纯化,接种家兔制备多克隆抗体。用PCR方法扩增PPV VP2全长基因,扩增产物克隆至表达载体pET-32a并转化至E.coli BL21(DE3)感受态中。分别用不同诱导时间、IPTG浓度、温度诱导表达VP2重组蛋白,经SDS-PAGE电泳切胶回收纯化目的蛋白。对家兔进行3次免疫后,采集血清制备抗体。PCR扩增得到1 740bp的VP2基因片段;构建原核表达载体pET-32a-VP2,经37℃,IPTG浓度1.0mmol/L诱导表达4h可得分子质量大小约为82ku目的蛋白;间接ELISA检测抗体效价可达1∶12 800;Western blot证明所制备的抗体能够有效的应用于PPV VP2抗原的检测。本研究成功构建了表达PPV VP2基因的原核载体,获得了VP2蛋白,制备了兔抗多克隆抗体,为建立检测PPV VP2蛋白ELISA方法奠定了基础。     

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.