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.     

大肠杆菌多重耐药调控基因marR的克隆及其原核表达

为获得MarR多克隆抗体,试验以大肠杆菌ATCC25922基因组为模板,根据GenBank中大肠杆菌的marR基因序列设计引物,PCR扩增出长约435bp的marR基因片段,将所得片段与pMD18-T载体连接,转化至JM109大肠杆菌中,筛选阳性克隆,其质粒中插入序列的测序结果与GenBank中报道一致,从阳性克隆中提取质粒,经BamHⅠ和XhoⅠ酶切回收435bp的目的片段,定向克隆到pET-28a(+)表达载体中,提取阳性质粒转化到大肠杆菌BL21(DE3)中获得阳性克隆。结果表明:经IPTG诱导阳性菌收集表达产物,通过SDS-PAGE分析证实marR基因得到表达;经Western-blot检测该蛋白具有良好的反应原性。     

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.     

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。     

溆浦鹅卵泡抑制素α亚基编码区cDNA克隆及其原核表达

根据GenBank中鸡卵泡抑制素α亚基序列设计引物，对该序列按大肠杆菌嗜好密码子进行优化，在设计的引物两端分别加上限制性内切酶SacⅠ和XhoⅡ的识别位点序列。利用RT-PCR技术从溆浦鹅卵泡的颗粒细胞总RNA中扩增出抑制素α亚基成熟区序列，经PCR扩增出354bp片段，该片段与pMD18-T载体连接，转化JM109感受态细胞，所得阳性克隆进行双酶切鉴定和PCR鉴定，并进行了测序分析，得到的克隆序列与设计的序列基本一致。表明成功地获得了抑制素α亚基编码序列的克隆载体。从阳性细菌中提取质粒，经SacⅠ和XhoⅠ酶切，回收354bp目的片段，定向克隆到pET28a中，转化DH5α受体菌，提取质粒，再转化到BL21(DE3)中，成功筛选出阳性克隆。阳性菌经IPTG诱导，通过SDS-PAGE，检测出溆浦鹅卵泡抑制素α亚基编码区的表达。     

牛抵抗素基因的克隆及原核表达

从健康新生牛大网膜脂肪组织中提取总RNA,根据已发表的牛Resistin mRNA序列设计、合成引物,通过RT-PCR进行cDNA扩增,获得了343 bp的片段。将该片段克隆于pMD18-T载体后进行序列分析,确认PCR产物为牛Resistin cDNA。从阳性克隆中提取质粒,经BamHⅠ和XhoⅠ双酶切,回收343 bp的目的片段,定向克隆到pGEX-6P-1表达载体中,提取质粒并再次转化到BL21(DE3)中,成功地筛选出阳性克隆。经IPTG诱导阳性菌,通过SDS-PAGE检测出牛的Resistin基因的表达。     

猪Ghrelin基因的克隆及原核表达

从猪下丘脑、胃等组织中提取总RNA，根据已发表的猪的Ghrelin mRNA序列设计合成引物，通过RT-PCR进行cDNA扩增，获得了282bp的片段。将该片段克隆于pMD-18T载体后进行序列分析，确认PCR产物为Ghrelin cDNA。从阳性克隆中提取质粒，经NheⅠ和XhoⅠ双酶切，回收282bp的目的片段，定向克隆到pET-28a表达载体中，提取质粒并再次转化到BL21（DE3）中，成功地筛选出阳性克隆。经IPTG诱导阳性菌，通过SDS-PAGE检测出猪Ghrelin基因的表达．     

枯草杆菌多重耐药基因bmr的克隆及原核表达

根据BenBank上已公布的多重耐药基因bmr的编码序列设计1对引物．以枯草杆菌基因组为模板，扩增出1170bp的DNA片段，将所得片段与pMD18T载体连接，转化到DH5α大肠杆菌中，成功地筛选到阳性克隆，其扩增片段测序结果与文献报道序列同源性达98．81％。将该片段定向克隆到pET-28a（＋）表达质粒中。提取质粒转化到BL21（DE3）中，经1mmol／l。IPTG诱导阳性菌．通过SDS-PAGE检测出bmr基因的表达产物。     

猪瘟病毒Erns基因的克隆及原核表达

从猪瘟病毒石门株血液样品中提取总RNA,通过R-PCR对Erns基因进行cDNA扩增,获得了696 bp的片段.将该片段克隆于T-easy载体后进行序列分析,确认PCR产物为猪瘟病毒Erns基因,从阳性克隆中提取质粒,经Bam H Ⅰ和Hin d Ⅲ双酶切,回收产物亚克隆到pET-32a表达载体中,提取质粒后转化BL21(DE3)感受态细胞,并筛选出阳性克隆,经IPTG诱导后通过SDS-PAGE检测出Erns基因的表达.     

牛干扰素-γ基因的RT-PCR扩增、序列分析及其在大肠杆菌中的表达

根据GenBank上发表的牛干扰素γ基因序列设计引物,从牛外周血淋巴细胞中提取基因组RNA,采用RTPCR技术,扩增出干扰素γ基因并进行序列分析。琼脂糖凝胶电泳显示牛干扰素γ扩增片段约为500bp。序列分析结果表明,牛干扰素γ基因序列全长517bp,开放阅读框架内501个核苷酸,共编码166个氨基酸,分子质量19.4ku,与参考序列完全一致。克隆序列经BamHⅠ/EcoRⅠ双酶切后连接到经同样双酶切的PBV220质粒中,转化入大肠杆菌DH5α中,筛选阳性克隆进行温度诱导表达,经SDSPAGE分析,在约19.4ku处有表达的蛋白带,表达产物经免疫荧光染色鉴定为阳性。     

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。     

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。     

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。     

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。     

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.