以杆状病毒为载体在昆虫细胞中表达致弱Ⅰ型马立克氏病病毒pp38基因同源物

用限制性内切酶Eco RI将不含起始密码子的pp38 基因从重组转移载体质粒pVLpp38 Ⅰ中切出,将致弱Ⅰ型MDV pp38 基因同源物克隆进该转移载体的相同位点。用所得的含pp38 基因同源物的重组转移载体质粒pVL pp38′与野生型杆状病毒(AcMNPV)以脂质体介导法共转染昆虫细胞系Sf9 后,用单克隆抗体H19 介导的间接荧光抗体法筛选到能表达MDV pp38 基因同源物的重组杆状病毒克隆。经SDS-PAGE和Western blot试验,证实pp38 基因同源物在Sf9 细胞中得到表达,抗重组杆状病毒血清能像抗MDV 血清那样,与MDV Ⅰ、Ⅱ、Ⅲ型病毒感染的鸡胚成纤维细胞在荧光抗体试验中呈阳性反应。     

禽多杀性巴氏杆菌外膜蛋白H基因在真核细胞中的表达及检测

利用PCR技术扩增出禽多杀性巴氏杆菌1 050 bp的外膜蛋白H基因(omph)片段,克隆到pMD18-T上,再亚克隆到真核载体pcDNA3.1(+)上,构建重组质粒pOMPH,将该重组质粒体外转染SP2/0细胞,检测其表达情况。结果表明本试验成功构建了真核表达质粒pOMPH,通过RT-PCR由转染了重组质粒的SP2/0细胞中扩增出了目的条带,Western blotting分析结果显示重组质粒pOMPH转染的SP2/0细胞泳道出现38ku的特异条带,间接免疫荧光分析表明在转染了pOMPH的SP2/0细胞中出现绿色荧光,说明omph基因在SP2/0细胞中成功表达了活性蛋白,从而为进一步研制DNA疫苗奠定了基础。     

表达鸡传染性喉气管炎病毒GB基因重组鸡痘病毒转移载体的构建

根据文献设计2对引物,PCR扩增FPV复制非必需区中外源基因插入位点两侧翼的片段,分别克隆到pUC19和pSK质粒中,并命名为pFP1和pFP2。根据文献设计引物,PCR扩增鸡传染性喉气管炎病毒(ILTV)的GB基因的编码区。将GB基因克隆到质粒pFP1中获得重组质粒pFP1-GB;用酶切质粒pEGFP,得到GFP片段并克隆到质粒pFP2中,得到重组质粒pFP2-GFP。双酶切pFP1-GB得到FP1-GB片段,并克隆到质粒pFP2-GFP中,得到重组质粒pFP1GB-FP2GPF。酶切质粒pE/L-7.5,获得背向连接的2个鸡痘病毒启动子片段——PE/L-7.5,并将该片段克隆到重组质粒pFP1GB-FP2GPF获得重组质粒pGB-GFP,该质粒即为鸡痘病毒的转移质粒。在质粒pGB-GFP中,2个背向连接的启动子被克隆到的GB和GFP基因之间,分别启动GB和GFP基因的表达。经酶切和测序鉴定pGB-GFP,启动子PE/L和P7.5已分别正确地插入到GB和GFP基因之间,分别启动外源表达基因GB和筛选标记基因GFP的表达,从而证明已获得表达鸡传染性喉气管炎病毒GB基因重组鸡痘病毒转移载体pGB-GFP。该载体为下一步获得表达鸡传染性喉气管炎病毒GB基因的重组鸡痘病毒奠定基础。     

火鸡疱疹病毒转移载体的质粒的构建

本试验采用以SV40早期启动子控制的大肠杆菌β—半乳糖苷酶(LacZ)基因作为报告基因,将其插入到含有火鸡疮疹病毒(HVT)糖蛋白A(gA)抗原基因的克隆片段中,构建成HVT转移载体质粒,在该转移载体质粒中,大肠杆菌LacZ基因的两端分别与两段1.4kb和1.8kb的HVT同源片段相连,克隆于pUC19质粒中,将含有LacZ基因的载体质粒与感染HVT的鸡胚成纤维细胞(CEF)中提取的全细胞DNA共同转染CEF,待病毒蚀斑出现后,用含有X-gal的琼脂糖覆盖细胞单层,可见到有兰色的病毒蚀斑,这些结果表明外源性的LacZ基因已被重组到HVT DNA中,同时还证明HVT的gA基因与HVT的复制无关,可以用于外源基因的插入和表达。     

Am80和TSA对鸡Stra8基因启动子活性的调控作用

旨在确定鸡Stra8基因启动子的主要调控区域,预测调控元件结合位点,探索用他米巴洛丁(Am80)或曲古抑菌素(TSA)诱导对Stra8启动子活性的调控作用。将鸡Stra8基因5′侧翼系列缺失片段插入到pGL3-basic载体构建重组质粒,并转染DF-1和GC-1,通过检测荧光素酶活性和预测启动子区域调控元件的结合位点,选取合适的启动子片段并构建其重组质粒Stra8-EGFP;将Am80和TSA分别按一定的浓度梯度诱导转染细胞,进行荧光素酶活性检测以筛选最佳诱导浓度;用最适剂量的Am80和TSA分别单独或联合诱导转染重组质粒Stra8-EGFP的P19,并检测各诱导组的绿色荧光表达程度。结果表明,鸡Stra8基因启动子-1 055~+54bp片段的活性较强,且含有RARα、RXRα和RARβ的结合位点;分别单独或联合使用Am80和TSA对转染的细胞进行诱导,结果显示,Am80和TSA协同作用的荧光素酶活性最高;重组质粒Stra8-EGFP转染细胞后,用Am80(10-5 mol·L-1)和TSA(10-6 mol·L-1)联合诱导可见绿色荧光强度最强。本研究成功分析了鸡Stra8基因启动子的活性和调控元件的结合位点,确定Am80和TSA共同诱导可显著提高Stra8基因启动子调控活性。     

ssc-miR-148a启动子克隆及其特征分析

为研究猪miR-148a(ssc-miR-148a)的转录调控机制,对其启动子进行了克隆及分析。本试验首先设计特异性PCR扩增引物,分别得到ssc-miR-148a前体上游3个片段,并将其连接到荧光素酶报告载体pGL3-Basic上。通过生物信息学方法,在线分析ssc-miR-148a启动子大概区域、甲基化部位和转录因子结合部位。将重组报告质粒转染293T细胞,分析启动子活性。采用不同浓度碱性成纤维生长因子(bFGF)处理猪成纤维细胞和转染有重组报告质粒的猪成纤维细胞,检测ssc-miR-148a和DNA甲基化转移酶1(DNMT1)的表达,及其对启动子活性的影响。结果显示,克隆得到的ssc-miR-148a启动子区2 043bp片段具有启动子活性,该序列存在5个CpG岛、Sp1及AP2等转录因子结合位点。0、5和10ng·mL-1浓度bFGF处理猪成纤维细胞和转染重组报告质粒的猪成纤维细胞后,ssc-miR-148a表达均显著下降(P0.05),DNMT1 mRNA显著增加(P0.05)。启动子活性均显著下降(P0.05),5和10ng·mL-1浓度间无显著差异(P0.05)。结果表明,ssc-miR-148a启动子位于前体上游2 043bp片段内,启动子区域有转录因子SP1结合位点,其表达受bFGF的调控。     

奶牛溶菌酶基因(Lyz)乳腺特异性表达质粒构建与鉴定

研究旨在构建具有高效抗菌、活性持久的奶牛乳腺特异性表达质粒,探索研制新型抗菌制剂,为利用基因工程技术防治奶牛乳房炎提供重要材料。根据GenBank中奶牛溶菌酶基因(lysozyme,Lyz)mRNA序列(GenBank号:NM_180999.1)设计引物,从奶牛乳腺上皮细胞中RT-PCR扩增Lyz基因编码序列,将其克隆到携带增强型绿色荧光蛋白通用型表达载体pEGFP-N1中测序。重组质粒pEGFP-N1-Lyz经Ase I+Hind III双酶切除CMV启动子,将奶牛乳腺特异性表达β-乳球蛋白基因(BLG)启动子同源重组替换到Ase I和Hind III酶切位点,构建奶牛溶菌酶基因(Lyz)乳腺特异性表达质粒pBLG-EGFP-N1-Lyz,采用脂质体法转染奶牛乳腺上皮细胞(BMEC)、人肾脏上皮细胞(HEK293T),荧光显微镜检测转染细胞中绿色荧光蛋白的表达情况。结果表明,重组表达质粒pBLG-EGFP-N1-Lyz测序结果与目的片段长度相符,转染至BMEC细胞和HEK293T细胞,在奶牛乳腺上皮细胞观察到绿色荧光, HEK293T细胞未观察到绿色荧光,奶牛Lyz基因乳腺特异性表达质粒构建成功。     

利用BrdU在CEF(TK~ )细胞中筛选TK~-重组鸡痘病毒

将新城疫病毒 (NDV)四平株 HN基因 ,插入不含报告基因、以 TK为侧翼的鸡痘病毒表达载体 p UTA- 2中的复合启动子下游 ,获得重组表达质粒 p UTA- 2 HN。将重组表达质粒转染鸡痘病毒 (FPV)感染的鸡胚成纤维细胞 (CEF) ,培养、收获病毒后 ,用含 40 m g/ L 5 -溴 - 2 -脱氧尿嘧啶 (Brd U )的培养液 ,在 CEF(TK )细胞中筛选培养 2代 ,然后用不含 Brd U的培养液进行病毒蚀斑纯化 ,成功筛选出表达 NDV HN蛋白的 TK-重组鸡痘病毒     

马立克氏病毒1.8-kb mRNA对其上游双向启动子活性的影响

构建了针对1.8-kb mRNA基因簇潜在阅读框的RNA干扰质粒pP(1.8-kb-RNAi).将该质粒与包含其上游双向启动子的质粒pP(pp38)-CAT和pP(1.8-kb)-CAT共转染鸡胚成纤维细胞(CEF)和MDV rMd5感染的CEF(rMd5-CEF),48 h后,通过测定转染细胞裂解液中氟霉素乙酰转移酶(CAT)的活性确定1.8-kb mRNA被干扰后对双向启动子活性的影响.结果显示,利用pP(1.8-kb-RNAi)质粒干扰1.8-kb mRNA,可以使其上游双向启动子两个方向的活性均显著下降(P<0.01),其中1.8-kb mRNA方向下跌29.5%,pp38方向下跌25.0%.本研究结果证明了1.8-kb mRNA对其上游双向启动子活性有增强作用.     

表达gpt和EGFP基因重组山羊痘病毒的构建和鉴定

在包含山羊痘病毒(Goatpox virus,GPV)疫苗株复制非必需区TK(Thymidine kinase)基因和背靠背启动子的质粒PtkPP的两个启动子下游插入EGFP(增强绿色荧光蛋白)基因,构建了质粒PtkPegfpP和PtkPPegfp,这两个重组质粒分别瞬时转染LT细胞,验证了启动子P11和P7.5能成功表达EGFP基因后,在PtkPP启动子P11的下游插入gpt(黄嘌呤鸟嘌呤磷酸转移酶)基因和EGFP基因,构建重组质粒PtkPgpt-egfpP,该重组质粒和GPV同源重组获得重组病毒rGPV-PtkPgpt-egfpP,利用MPA(霉芬酸)阻断核酸代谢途径筛选到稳定表达EGFP基因的重组GPV。为进一步开展GPV活载体疫苗的研究提供了技术平台。     

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.