胸膜莫肺炎放线杆菌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AppDNA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39.5KDa。利用HiTrapFFcrude columns将表达的蛋白进行了纯化。     

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。     

旋毛虫T3223-6 cDNA的表达及鉴定

利用PCR技术将T3223-6 cDNA扩增克隆到原核表达载体pET-28a,将重组质粒转入克隆菌Nova-blue,提取质粒进行酶切和测序鉴定后转入表达菌BL21star(DE3).用1 mM IPTG诱导培养重组表达菌,对菌体裂解物进行SDS-PAGE分析,检测重组蛋白的表达情况.用旋毛虫感染猪血清和正常血清,通过western blotting检测重组蛋白的反应原性.结果表明:经IPTG诱导后重组转化菌的裂解产物出现44 kD左右的表达带,大小与理论值相符;western blotting检测结果显示重组蛋白可以被旋毛虫感染猪血清识别,具有反应原性.     

鼠脱氧核糖核酸酶（DNaseⅡα）基因的克隆及原核表达

根据鼠脱氧核糖核酸酶Ⅱ (DNase Ⅱ)基因设计引物,通过RT-PCR 扩增出鼠DNase Ⅱα基因片段,克隆到pMD-18T载体,然后转化至大肠杆菌JM109.经鉴定及序列测定,克隆基因与鼠DNase Ⅱα基因序列完全一致,大小为1 008 bp.将重组质粒pMD-DNase Ⅱα进行酶切后连接到原核表达载体pET-28a中,重组质粒经PCR和双酶切鉴定及序列分析正确后转化到大肠杆菌BL21(DE3)中,IPTG诱导表达,表达产物经SDS-PAGE检测,然后进行初步纯化,Western-blot鉴定.结果表明,本试验成功构建了pET-28a-DNase Ⅱα表达载体;表达产物以包涵体形式存在,相对分子质量约为38 800;初步纯化的目的蛋白在SDS-PAGE图谱上呈单一条带,Western-blot鉴定能与抗体发生特异免疫反应.     

鸡柔嫩艾美耳球虫ZJ株TA4基因的克隆及原核表达

本试验对E．tenella ZJ株的TA4基因进行了克隆和表达。根据已报道的柔嫩艾美尔球虫TA4基因序列设计引物,以孢子化卵囊总RNA为模板,用RT—PCR的方法扩增得到一特异片段,将扩增产物克隆至pMD18-T,转化DH5-α感受态,经酶切鉴定获得阳性重组质粒并对其进行测序。测序结果与参考序列比较,核苷酸同源性为99．1％。然后将重组质粒和表达载体pGEX-4T2分别以EcoR I、Xho I酶切后构建重组表达载体pGEX—TA4,并将其转化入大肠杆菌BL21中,提取质粒经酶切和PCR鉴定正确后,用IPTG诱导表达。表达产物经SDS—PAGE和Western blot检测显示,TA4基因在大肠杆菌中获得表达;融合蛋白的分子量约为43ku,以包涵体形式存在;诱导6h的蛋白表达量可达到35.9％,采用抗GST抗体进行Western blot,成功检测到了该特异性条带。     

鸡柔嫩艾美尔球虫ZJ株3-1E基因的原核表达及鉴定

本实验对E．tenellaZJ株的3-1E基因进行了克隆和表达。根据已报道的柔嫩艾美尔球虫3-1E基因序列设计引物,以孢子化卵囊总RNA为模板,用RT—PCR方法扩增得到一条特异片段,将扩增产物克隆至pUCM—T,转化感受态菌DH5仅,经酶切鉴定获得阳性重组质粒并对其进行测序。测序结果与参考序列比较,核苷酸同源性为99．5％。然后将重组质粒和表达载体pET-30a分别以EcoRⅠ、SalⅠ酶切后构建重组表达载体pET-30a-3—1E,并将其转化入大肠杆菌BL21中,提取质粒经酶切和PCR鉴定正确后,用IPTG诱导表达。表达产物经SDS—PAGE和Westernblot检测显示,3-1E基因在大肠杆菌中成功表达;融合蛋白的分子量约为27ku,诱导6h的蛋白表达量可达到47、024％。     

鸡柔嫩艾美耳球虫ZJ株5401基因在大肠杆菌中的表达

根据E．tenella子孢子表面抗原5401基因序列设计合成特异性引物，引物两端分别加上EcoRⅠ和SalⅠ酶切位点及保护碱基，用RT-PCR方法从E．tenella孢子化卵囊扩增出881bp的片段。将重组克隆质粒pGEM-T5401用EcoRⅠ和SalⅠ双酶切后，电泳回收目的片段，克隆到同样经EcoRⅠ和SalⅠ双酶切的表达载体pET-30a中，得到重组表达质粒pET-30a-5401，把重组表达质粒转化大肠杆菌BL21，经IPTG诱导，表达出了His-5401融合蛋白。Western印迹结果表明表达产物为大约66．2ku的蛋白。     

微小隐孢子虫类钙调蛋白基因的克隆与原核表达

为了对微小隐孢子虫(C.parvum)类钙调蛋白(CML)基因进行原核表达,分析重组表达蛋白的反应原性。以C.parvum卵囊cDNA为模板,用PCR方法扩增得到C.parvum CML基因。将CML基因连接到克隆载体pMD18-T,获得重组质粒pMD-CML,经限制性内切酶BamHⅠ和XhoⅠ双酶切后,连接到经相同内切酶酶切的表达载体pGEX-6p-1上,构建重组表达质粒,转化到大肠埃希菌BL21(DE3)中进行诱导表达。利用GST亲和树脂法纯化重组表达蛋白,对纯化的重组蛋白进行Western blot分析。结果表明,成功构建了重组原核表达质粒pGEX-CML,重组质粒转化菌经IPTG诱导后成功地表达出了分子质量约为51ku的重组蛋白rCML,纯化的蛋白rCML能与感染兔隐孢子虫(C.cuniculus)的兔血清发生特异性反应,具有很好的反应原性。     

仙台病毒P基因的原核表达及抗原性分析

根据GenBank公布的仙台病毒(SeV)磷蛋白(P)基因序列,设计合成一对特异性引物,PCR扩增P基因,并将其克隆至原核表达载体pET-32a中;重组质粒经酶切、PCR及测序鉴定正确后.转化感受态细胞BL21(DE3)PlysS,经IPTG诱导表达,获得相对分子量为99.6 ku的融合蛋白.经western blot及ELISA分析表明表达的融合蛋白具有良好的抗原性.     

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.