猪轮状病毒OSU强毒株vp7基因的克隆及其抗原表位原核表达和免疫学活性分析

参照GenBank中所收录的猪轮状病毒OSU株序列设计了一对特异性引物,RT—PCR扩增出1062bp的vp7全长基因,与参考OSU序列的核苷酸同源性达99．8％,克隆到pMD18-T载体中,获得pMD18-T—vp7阳性质粒。以阳性质粒为模板,利用引入BamH Ⅰ和XhoⅠ酶切位点的引物进行PCR扩增得到含有vP7中和抗原表位区域639bp的基因片段,定向克隆到表达载体pGEX-6P—1中,经酶切PCR鉴定的阳性质粒转化大肠杆菌BL21（DE3）,经IPTG（终浓度为1．0mmol／mL）37℃诱导4h后,SDS—PAGE电泳显示表达了大小约50ku带GST标签的融合蛋白,薄层扫描显示蛋白表达融合量占菌体总蛋白的33．2％。Westernblot分析表明该VP7重组蛋白可与PRV阳性血清特异性反应。     

家兔Ⅰ型肽载体(PepTⅠ)基因的克隆与原核表达

用real-time PCR方法扩增家兔PepTⅠmRNA序列,通过原核表达体外获得家兔PepTⅠ蛋白,旨在制备多克隆抗体。试验通过RT-PCR方法成功扩增出2 001bp的家兔PepTI基因片段,设计特异性引物成功扩增出抗原表位相对集中的1 071bp大小的片段,构建重组表达质粒,获得融合蛋白。结果显示:家兔PepTⅠ基因片段成功的连接到pMD18-T载体上,重组克隆载体双酶切后回收目的片段分别连接到原核表达载体pET-32a、pET-28a,经转化提取质粒测序验证后表明,成功构建原核表达载体pET-32a-P、pET-28a-P,将重组质粒转化至感受态细胞BL21(DE3)后IPTG诱导,成功表达出PepTⅠ融合蛋白,目的蛋白相对分子质量大小约为44 000,与预期试验结果相符。结果表明:本试验成功扩增出家兔PepTⅠmRNA序列,并且通过原核表达获得家兔PepTI融合蛋白。     

牛瑟氏泰勒虫HSP70基因的克隆及原核表达

根据GenBank(D12692.1)上发表的牛瑟氏泰勒虫HSP70基因序列设计、合成1对特异性的引物,采用PCR方法扩增牛瑟氏泰勒虫HSP70基因片段,将扩增产物与pMD18-Tsimple载体连接,重组质粒经PCR、酶切鉴定后测序;用pGEX-4T-1表达载体构建重组质粒pGEX-HSP70,经IPTG诱导表达后进行SDS-PAGE、Western-blot分析。结果表明:扩增的HSP70基因与D12692.1序列同源性为99.3%;表达的融合蛋白分子质量约为68ku,而且可与牛瑟氏泰勒虫阳性血清发生特异性反应。     

秦川牛生长抑制素基因克隆及原核表达研究

 试验旨在通过基因工程方法获得重组肌肉生长抑制素蛋白。提取秦川牛的骨骼肌总RNA,根据基因库中海福特牛肌肉生长抑制素基因序列设计合成特异性引物,引物两端分别加上Bam HⅠ和Xho Ⅰ酶切位点及保护碱基,用RT-PCR方法扩增肌肉生长抑制素全长基因,并将其克隆到pMD18-T载体上,测序后经Bam HⅠ和Xho Ⅰ双酶切,将目的片段插入到PGEX-4T-1中,构建原核表达载体PGEX-4T-1-M,将重组表达质粒转化至Rosetta(DE3)中诱导表达。结果表明,扩增的秦川牛肌肉生长抑制素全长基因1 128 bp,克隆载体经过DNA序列测定,所得基因与GenBank上发表的一致;成功构建原核表达载体PGEX-4T-1-M,经IPTG诱导,表达出了GST-M融合蛋白,用GST标签抗体做Western blotting印记证明产物大约69 ku,与预期大小相符,并在Rosetta（DE3）菌中成功的进行融合蛋白表达。     

PRV SA株gB基因和EGFP基因在BHK细胞中的融合表达研究

为了研究gB蛋白在细胞内的作用位点,试验以猪伪狂犬病病毒(Pseudorabies virus,PRV)SA株为模板,设计特异性引物,扩增gB基因保守片段;以pEGFP-N1质粒为模板扩增EGFP基因;以gB基因与EGFP基因胶回收产物为模板,用gB基因上游引物及EGFP基因下游引物通过重叠延伸PCR(SOE PCR)技术获得gB/EGFP融合基因;构建pcDNA3.1/gB/EGFP重组表达质粒,转染乳仓鼠肾细胞(BHK细胞),采用Western-blot技术及荧光显微镜检测荧光蛋白在细胞内的表达情况。结果表明:试验成功扩增到300 bp的gB基因、760 bp的EGFP基因及1 060 bp的gB/EGFP融合基因;成功构建出pcDNA3.1/gB/EGFP重组表达质粒并转染BHK细胞;Western-blot技术及荧光显微镜检测显示,融合蛋白在BHK细胞中成功表达。说明通过荧光蛋白研究目的蛋白的作用位点是可行的。     

李氏杆菌溶血素基因的表达及活性分析

根据LMO溶血素基因hlyA设计引物,PCR扩增hlyA,将扩增产物与pMD18-T连接,重组质粒经酶切鉴定、PCR分析以及确证性测序。将由重组质粒pMD18-hlyA上扩增的缺失信号肽序列的目的片段与表达载体DGEX-4T-1分别酶切连接后,转化BL21细胞。筛选阳性克隆,用IPTG诱导表达,SDS-PAGE和Western blot分析,纯化重组融合蛋白进行溶血试验、小鼠致病力试验和间接ELISA分析。结果表明hlyA基因在大肠杆菌中成功表达分子量82Ku的融合蛋白,能裂解红细胞,且能被LMO阳性血清所识别。一定剂量腹腔注射能将小鼠致死。以此为包被抗原的间接ELISA可以将LMO阴、阳性血清分开。这表明GST-LLO融合蛋白具有良好的生物活性,可用于李氏杆菌病的间接ELISA诊断。     

产单核细胞李斯特菌溶血素基因的克隆及原核表达

利用PCR方法扩增产单核细胞李斯特菌（LM）的溶血素基因hlyA,获得一条大小约为1600bp的目的片段,将其与pMD18-T载体连接,经酶切、PCR鉴定,命名为pMD18-T—hlyA。测序结果显示所扩增的hlyA基因与GenBank中发表的hlyA的序列同源性为99％。利用含有BamHⅠ／XhoⅠ酶切位点引物B从pMD18-T—hlyA扩增不含信号肽序列的目的片段B,获得了大小约为1500bp的基因片段,该片段与pET32a表达载体经BamHⅠ／XhoⅠ处理后进行连接,转化BL21受菌体。通过酶切、PCR鉴定及序列测定后获得阳性pET32a-hlyA表达重组质粒,将重组菌用终浓度为0．1mmol／L的IPTG进行诱导表达,结果表明重组菌在诱导2h～3h表达量最高,融合蛋白约占菌体的40％,分子量大小约为80ku;利用LM的阳性血清进行Western blot分析,证实该融合蛋白可以被LM阳性血清所识别,为今后研制针对该蛋白的单克隆抗体和建立间接ELISA诊断方法提供了条件。     

猪繁殖与呼吸综合征病毒SCQ株M蛋白基因截断片段原核表达载体构建及其表达的初步研究

为表达猪繁殖与呼吸综合征病毒(PRRSV)M蛋白主要抗原表位基因序列,参照GenBank中发表的PRRSV SCQ株M蛋白基因设计并合成一对特异性引物,通过PCR方法从重组质粒pMD18-T-M扩增得到缺失N端跨膜区的M蛋白基因片段dM(deleting M),将其与pMD19-T simple vector连接,经测序正确后克隆至高效原核表达载体pGEX-4T-1,得到重组表达载体pGEX-4T-1-dM,并将其转化大肠杆菌Rosetta(DE3),经IPTG于37℃诱导,PRRSV M基因获得表达。经SDS-PAGE分析,所表达的融合蛋白分子量约为35 kDa。以纯化的重组蛋白作为抗原,经Western Blot分析结果表明该重组蛋白可被PRRSV阳性血清所识别,可用于PRRSV的检测。     

鸽圆环病毒浙江株△Cap基因的克隆与原核表达

根据已发表的鸽圆环病毒(PiCV)序列,在V1ORF保守序列部位设计引物(目的片段长度为629bp),对浙江某鸽养殖场的病料进行PCR扩增检测,获得阳性样品;应用设计的删除核定位信号外壳蛋白基因(△Cap)的引物,扩增获得680bp的△Cap基因,克隆于pMD18-T载体,进行测序;将△Cap基因亚克隆到原核表达载体pET-28a进行融合表达,SDS-PAGE电泳检测发现,△Cap融合蛋白经IPTG诱导后在大肠杆菌中以包涵体形式表达,目的蛋白表达量占菌体总蛋白的22.1%。     

猪繁殖与呼吸综合征病毒SGQ株M蛋白基因截断片段原核表达载体构建及其表达的初步研究

为表达猪繁殖与呼吸综合征病毒（PRRSV）M蛋白主要抗原表位基因序列,参照GenBank中发表的PRRSVSCQ株M蛋白基因设计并合成一对特异性引物,通过PCR方法从重组质粒pMD18-T-M扩增得到缺失N端跨膜区的M蛋白基因片段dM（deletingM）,将其与pMD19-Tsimplevector连接,经测序正确后克隆至高效原核表达载体pGEX-4T-1,得到重组表达载体pGEX-4T-1-dM,并将其转化大肠杆菌Rosetta（DE3）,经IPTG于37℃诱导,PRRSVM基因获得表达。经SDS-PAGE分析,所表达的融合蛋白分子量约为35kDa。以纯化的重组蛋白作为抗原,经WesternBlot分析结果表明该重组蛋白可被PRRSV阳性血清所识别,可用于PRRSV的检测。     

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.