鸡抗病毒Mx蛋白基因在毕赤酵母GS115中的表达

为了利用酵母表达技术制备鸡抗病毒Mx蛋白,试验采用基因工程技术,将编码鸡抗病毒Mx蛋白基因亚克隆至含有分泌信号肽序列的毕赤酵母表达载体中,构建成分泌型重组表达载体pPIC9K-Mx;用电转化法将线性化的pPIC9K-Mx转化至毕赤酵母菌株GS115中,G418梯度筛选转化菌;再经MM与MD平板对比生长试验筛选,PCR鉴定目的片段,筛选出高拷贝重组子,该高拷贝菌株分别经0.5%、1%甲醇诱导,表达产物再经SDS-PAGE检测。结果表明:成功构建了鸡抗病毒Mx蛋白基因的毕赤酵母表达载体,经G418抗性筛选得到高拷贝菌株;在甲醇含量维持在1%时,鸡抗病毒Mx蛋白在毕赤酵母GS115中获得良好表达,表达产物大小约为75ku;表达蛋白约占表达上清液的39.2%。     

猪源抗菌肽PBD-1在毕赤酵母中的表达及鉴定

本试验旨在实现猪β防御素1 (poricine-β-defensin 1,PBD-1)在毕赤酵母中的表达,获得有抗菌活性的抗菌肽PBD-1。根据PBD-1的氨基酸序列和酵母密码子偏好性,设计优化其核苷酸序列,利用SOE-PCR技术获得PBD-1基因序列,克隆到毕赤酵母表达载体pPIC9K中,构建重组质粒pPIC9K-PBD-1,经SacⅠ线性化后转入毕赤酵母SMD1168中。PCR筛选得到阳性酵母表达菌株,经甲醇诱导后得到分子质量约4.5 ku的抗菌肽PBD-1。抗菌特性研究结果表明,表达产物抗菌肽PBD-1对大肠杆菌、金黄色葡萄球菌及枯草芽孢杆菌均有较好的抑制效果。     

中国林蛙抗菌肽基因在毕赤酵母中的表达及其活性检测

将中国林蛙皮肤抗菌肽基因(RC)克隆到毕赤酵母表达载体pPIC9K上,使之准确融合于а交配因子分泌信号,然后通过电击转化毕赤酵母宿主菌GS115/His-,构建pPIC9K/RC。将筛选出的高效表达的重组转化子用甲醇作诱导剂进行小瓶发酵,28～30℃诱导后,经Tricine SDS-PAGE检测,表达产物在а信号因子引导下分泌到培养基中。分泌到培养基中的表达产物能够抑杀细菌和抑制肿瘤细胞生长。对酵母重组子用酵母染色体DNA的通用引物和目的片段的引物进行PCR扩增。结果表明,中国林蛙皮肤抗菌肽基因能以单拷贝整合到毕赤酵母染色体基因组中并形成转录产物。     

H1N1猪流感病毒HA基因在毕赤酵母中的表达

利用RT-PCR扩增H1N1亚型猪流感病毒HA基因,亚克隆到毕赤酵母表达载体pPIC9K中,构建分泌型重组表达载体pPIC9K-HA。将线性化的pPIC9K-HA电转化毕赤酵母菌GS115。将经MD平板筛选和PCR鉴定的阳性菌株用G418筛选多拷贝重组子。最后用1%甲醇诱导表达重组子,经SDS-PAGE和Western-blot检测,H1N1亚型猪流感病毒HA基因在毕赤酵母中成功得到了表达,产物约60.4KD,并具有免疫活性。本研究为进一步研究猪H1N1亚型猪流感病毒HA基因的功能及检测方法奠定了基础。     

猪蛔虫抗菌肽Cecropin P2 基因的真核表达及表达产物的抗菌活性研究

为获得猪蛔虫抗菌肽Cecropin P2基因并研究其抗菌功能,根据CecropinP2成熟肽的编码基因,人工合成3条寡聚核苷酸片段,经PCR获取Cecropin P2基因序列,成功构建了重组表达质粒pPIC9 K-Cecropin P2-His6并转化毕赤酵母GS115感受态细胞。转化子经MD、MM以及G418筛选,1%甲醇诱导表达,利用Tris tricine-SDS-PAGE对表达产物进行检测。体外抑菌试验表明,获得的重组蛋白对大肠杆菌和金黄色葡萄球菌均有较好的抑菌活性。本试验为抗菌肽的应用和新抗菌肽类药物的开发提供了必要的试验基础。     

E.tenella河北株EtMIC-2基因的克隆及毕赤酵母系统表达

为表达鸡柔嫩艾美耳球虫(E.tenella)河北株EtMIC-2蛋白,本试验采用RT-PCR方法克隆出E.tenella河北株EtMIC-2基因,连接到毕赤酵母分泌表达载体pPIC9中,构建重组表达载体pPIC9-EtMIC-2。将其线化后转化到巴斯德毕赤酵母GS115中,甲醇诱导表达,饱和硫酸铵4℃沉淀浓缩后,用His选择镍-亲和层析柱纯化EtMIC-2蛋白。采用SDS-PAGE和Western blot方法验证其蛋白的表达。结果表明,构建的重组表达载体pPIC9-EtMIC-2在毕赤酵母菌中表达出相对分子质量约为45 000的目的蛋白,表达的EtMIC-2蛋白能被E.tenella阳性血清识别,具有良好的免疫反应原性。为进一步研究EtMIC-2蛋白功能及构建DNA疫苗奠定基础。     

牛α干扰素在毕赤酵母中的高效分泌表达及活性鉴定

为高效分泌表达牛α干扰素(boIFN-α),本研究通过人工合成boIFN-α基因,将目的基因克隆至表达载体pPIC9K中,构建重组质粒pPIC9K-boIFN-α,将其电转化于毕赤酵母菌株GS115,利用抗药选择压力G418筛选重组菌,对重组菌诱导表达,取上清进行SDS-PAGE和western blot检测,并优化重组菌的诱导表达条件.结果显示:筛选获得高效分泌表达boIFN-α的重组菌,其最佳诱导条件为:250 r/min,26℃培养,1％甲醇浓度诱导,诱导72 h.上清中目的蛋白表达量最高可达200 μg/mL,本研究为boIFN-α在生产中的应用奠定了基础.     

绵羊干扰素tau在毕赤酵母中的表达

应用绵羊的干扰素tau基因IFNt,根据毕赤酵母(Pichia pastoris)密码子选择偏好进行分子改造。改造后的基因IFNt克隆到毕赤酵母表达载体p PIC9上,构建得到重组毕赤酵母表达质粒p PIC9-IFNt。电转化于毕赤酵母GS115菌株,对转化后菌体诱导表达,上清SDS-PAGE检测,并优化诱导表达条件。结果表明:IFNt基因已重组到酵母染色体上,并能正常分泌表达,最佳诱导条件为:200 r/min,28℃,0.5%甲醇,96 h。该研究为真核表达绵羊IFNt的生产应用提供了一定的理论依据。     

重组猪蛔虫抗菌肽cecropin P1在毕赤酵母中的表达及抑菌活性

根据已发表的蛔虫抗菌肽cecropin P1基因编码序列设计引物,采用RT-PCR方法从猪肠道蛔虫中扩增出cecropin P1基因,并将其插入到酵母分泌型表达载体pPIC9K中α-因子信号肽下游的SnaBⅠ和NotⅠ位点之间,构建成cecropin P1基因的酵母分泌型表达载体pPIC9K/cecropin P1。载体经SalⅠ酶切线性化,电转化到组氨酸缺陷型的酵母宿主菌GS115中,然后利用以葡萄糖为碳源的培养基（MD）和以甲醇为碳源的培养基（MM）筛选出组氨酸His＋型和甲醇利用正型（Mut＋）酵母重组体,再经G418加压筛选得到高拷贝cecropin P1基因的重组酵母。经PCR检测证明cecropin P1基因已整合到毕赤酵母的染色体中。重组酵母经摇瓶发酵培养和甲醇诱导,通过Tricine-SDS-PAGE检测,重组酵母能够分泌表达重组cecropin P1,同时表达的cecropin P1对大肠杆菌和金黄色葡萄球菌均有明显的抑菌活性。     

猪带绦虫未知基因SLC10在毕赤酵母中的表达

SLC10是以反式剪切引导序列为基础,通过RT-PCR从猪囊尾蚴中克隆到的一个未知基因。将猪囊尾蚴SLC10基因从重组克隆载体pGEM-SLC10扩增并酶切后,与相应酶切处理的毕赤酵母分泌性表达载体pPIC9K相连接,构建重组表达载体pPIC9K-SLC10,转化大肠杆菌JM109,经PCR、酶切和测序鉴定,基因序列完全正确。纯化的重组质粒pPIC9K—SLC10用内切酶Sac I线性化,电转化毕赤酵母,使重组表达载体与酵母染色体发生同源整合;采用G418抗性梯度法筛选多拷贝重组菌株,用甲醇进行诱导表达;通过SDS-PAGE和Western—blot分析表达产物,结果表明目的蛋白得到了表达,但不具有免疫反应性。     

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.