产气荚膜梭菌α—β融合基因的高效表达

用PCR从含产气荚膜俊菌α毒素基因的质粒pXETA1中扩增出α毒素基因，用NcoⅠ和BamHI双酶切该α毒素基因，回收0．95kb的α毒素基因片段，再用NcoⅠ和BamHI双酶切含产气荚膜梭菌β毒素基因质粒pXCPAB2，与 回收的α毒素基因片段连接，转化至受菌BL21（DE3）中。经NcoI,BamHI,NcoI酶反应鉴定和核苷酸序列分析证实，获得了理想重组质粒pXCPAB2，该重组质粒含有α-β融合基因。重组菌株BL21（DE）3（pXCPAB2)经IPTG诱导后，其表达产物经ELISA检测和SDSPAGE分析，结果表明重组菌株可以高效表达α-β融合基因，该融合蛋白占菌体总蛋白的22．14％。     

表达产气荚膜梭菌α—β融合蛋白基因工程菌株的构建

用PCR从含产气荚膜俊菌α毒素基因的质粒pXETA1中护增出α毒素基因，用NcoI和BamHI双酶切该α毒素基因，回收0.95kb的α毒素基因片段，再用NcoI和BamHI双酶切含产气荚膜梭菌β毒素基因质粒pXETB2，与上述回收的α毒素基因片段连接，转化至受体菌BL21（DE3）中。经NcoI、BamHI、NotI酶切反应鉴定和核苷酸序列分析证实，获得了理想重组质粒pXCPAB2，该重组质粒含有α-β融合基因。重组菌株BL21（DE3）（pXCPAB2）经IPTG诱导后，其表达产物经ELISA检测和SDS-PAGE分析，结果表明重组菌株可以高效表达α-β融合蛋白，该融合蛋白占菌体总蛋白的22.14%。用从IPTG诱导的工程菌中提取的包涵体或经甲醛灭活的工程菌制成抗原免疫小鼠，免疫小鼠至少能抵抗2MLD的C型产气荚膜梭菌的攻击。这表明构建的工程菌株BL21（DE3）（pXCPAB2)可以作为预防仔猪红痢基因工程菌苗的候选株。     

产气荚膜梭菌α毒素保护性抗原基因的克隆与核苷酸序列分析

将含产气荚膜梭菌α毒素基因的质粒ｐＫＭＡ１００用限制性核酸内切酶ＢａｍＨＩ和ＨｉｎｄⅢ双酶切,经１．５％琼脂糖凝胶电泳分离、透析袋电洗脱法回收０．９５ｋｂα毒素基因片段,再将载体ｐＥＴ－２８ｂ（＋）用ＢａｍＨＩ和ＨｉｎｄⅢ双酶切,然后将处理好的ｐＥＴ－２８ｂ（＋）与０．９５ｋｂ的α毒素基因片段通过Ｔ４ＤＮＡ连接酶进行粘性末端连接,转化至受体菌ＢＬ２１（ＤＥ３）中。经ＢａｍＨＩ和ＨｉｎｄⅢ双酶切分析和核苷酸序列分析,证明重组质粒ｐＸＥＴＡ１含有产气荚膜梭菌α毒素基因。确定了α毒素基因的全部核苷酸序列,并证明其具有正确的阅读框架。     

产气荚膜梭菌β-毒素基因的克隆与核苷酸序列分析

为了给产气荚膜梭菌（Ｃｌｏｓｔｒｉｄｉｕｍｐｅｒｆｒｉｎｇｅｎｓ）β毒素基因工程亚单位苗和细菌毒素多价基因工程苗的研制提供基因材料，用聚合酶链式反应（ＰＣＲ）技术，从Ｂ型产气荚膜梭菌染色体基因组中扩增了９３０ｂｐ的β－毒素基因。用限制性核酸内切酶ＢａｍＨＩ和ＥｃｏＲＩ对ＰＣＲ产物进行双酶切处理，然后通过Ｔ４ＤＮＡ连接酶将其定向连接于事先经同样的双酶切处理的载体质粒ｐＥＴ－２８Ｃ（＋）的多克隆位点，转化至受体菌ＢＬ２１（ＤＥ３）中。经ＢａｍＨＩ和ＥｃｏＲＩ双酶切分析和ＰＣＲ扩增检测，证明重组质粒ｐＥＣＢ２中含有产气荚膜梭菌的β－毒素基因。经核苷酸序列分析，明确了克隆的β－毒素基因在重组质粒中的连接向位和阅读框架是正确的。     

CPA-LTB融合基因的构建与表达

利用PCR技术，从A型产气英膜梭菌染色体和pEWD299中分别扩增出a毒素（CPA）基因和LTB基因，通过分离、纯化、内切酶酶切、连接和转化，构建了含CPA—LTB融合基因表达质粒的重组菌株BL21（DE3）（pCPA—LTB）。经酶切鉴定和核苷酸序列测定证实，构建的重组质粒pET—CPA含有CPA—LTB融合基因，其基因序列和阅读框架均正确。经EISA检测和SDS—PAGE分析，重组菌株表达的CPA—LTB融合蛋白能够被α、LT毒素抗体所识别。重组菌株BL21（DE3）（pCPA—LTB）经IPTG诱导后，融合蛋白CPA—LTB的表达量占菌体总蛋白相对含量的12.5％。     

产气荚膜梭菌β-毒素基因的克隆及CPB-ST融合基因的构建

用聚合酶链式反应（ＰＣＲ）技术,从Ｂ型产气荚膜梭菌染色体基因组中扩增了９３０ｂｐ的β－毒素基因。用限制性核酸内切ａｍＨ Ⅰ和ＥｃｏＲ Ⅰ对ＰＣＲ产物进行双酶切处理,然后,通过Ｔ４ ＤＮＡ连接酶将其定向连接于事先经同样的双酶切处理的载体质粒ｐＥＴ－２８Ｃ（＋）的多克隆位点,转化至受体菌ＢＬ２１（ＤＥ３）中。经ＢａｍＨ Ⅰ和ＣｃｏＲ Ⅰ双酶切分析和ＰＣＲ扩增检测。证明重组质粒ｐＥＣＢ２中含有产气荚膜梭     

C型产气荚膜梭菌β毒素基因克隆与核苷酸序列分析

用ＰＣＲ技术,从含Ｃ型产气荚膜梭菌β毒素基因质粒ｐＢ１２中扩增出了０．９５Ｋｂ的β毒素基因,然后用限制性核酸内切酶ＢａｍＨＩ和ＥｃｏＲＩ对其进行双酶切处理,最后将其定向连接在事先经同样内切酶处理的载体ｐＥＴ－２８ｃ（＋）中的相应位点上,转化至受体菌ＢＬ２１（ＤＥ３）中。经ＢａｍＨＩ和ＥｃｏＲＩ双酶切分析和核苷酸序列分析,证明重组质粒ｐＸＥＴＢ２含有产气荚膜梭菌β毒素基因,确定了其全部的核苷酸序列,并且具有正确的阅读框架     

产气荚膜梭菌贵州分离株α-毒素基因的克隆与鉴定

提取了A型产气荚膜梭菌贵州分离株的染色体DNA，经聚合酶链反应(PCR)扩增并回收获得了1条242bp的特异性片段，将该基因片段克隆到质粒载体pUC19中，并转化至大肠埃希氏菌JM109中；经氨苄青霉素(Amp)抗药性和显色反应筛选及重组质粒酶切分析、PCR鉴定和Southern-blotting检测，表明该重组质粒是产气荚膜梭菌α-毒素基因的克隆。     

产气荚膜梭菌B型C58-1株β1毒素基因的克隆表达

利用PCR技术,从B型产气荚膜梭菌中国标准株C58-1株扩增出β1毒素基因,连接pMD18-T 载体筛选阳性克隆,然后用限制性核酸内切酶BamHⅠ和SalⅠ对其进行酶切,回收927 bp的β1毒素基因片段,将其定向克隆到载体pET-32a中,获得重组质粒pETβ927。将pETβ927转化至受体菌BL21(DE3)中,其表达产物经His-Trap FF预装柱纯化、SDS-PAGE 检测目的蛋白大小和分布及Western blotting检测其反应原性。结果表明,完整的β1毒素基因大小为1 011 bp,与GenBank发表的B型和C型产气荚膜梭菌β1毒素蛋白序列同源性达99.4%以上;SDS-PAGE结果显示重组目的蛋白在大肠杆菌中成功表达,融合蛋白大小为 54 ku,在超声波裂解上清和包涵体中均有分布,但以包涵体为主。Western blotting检测结果显示表达的重组蛋白可与特异性血清抗体发生免疫反应,表明β1毒素蛋白具有较好的反应原性。     

重组产气荚膜梭菌plc毒素基因植物乳酸杆菌的构建

依据乳酸菌的肠道益生作用,选择产气荚膜梭菌关键致病因子α毒素/磷脂酶C为抗原,构建产气荚膜梭菌α毒素去除信号肽的plc基因片段重组植物乳杆菌,利用植物乳酸菌穿梭载体pSIP409构建重组质粒pSIP409-plc,经双酶切鉴定和序列测定正确后转化大肠杆菌BL21(DE3)感受态细胞并进行SppIP诱导表达。Western Blot证明重组蛋白表达成功,并且主要以包涵体形式存在,plc重组蛋白相对分子质量分别为40 kDa。重组质粒pSIP409-plc分别电转化植物乳杆菌NC8细胞,PCR和双酶切鉴定正确后进行SppIP诱导表达。Western Blot和间接免疫荧光鉴定表明,构建的重组植物乳酸杆菌具有诱导分泌plc蛋白的能力,可作为黏膜免疫的候选抗原。     

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.