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为获得具有天然活性的猪传染性胃肠炎病毒(TGEV)重组S蛋白,制备针对猪传染性胃肠炎S蛋白A、D抗原位点的单克隆抗体及建立快速抗体检测方法,本研究将TGEV S基因A、D抗原位点经PCR扩增并克隆入p Fast Bac HBM-TOPO载体,经转座、转染后获得重组杆状病毒,并对重组杆状病毒进行间接免疫荧光(IFA)及Western blot分析,结果显示,TGEV S基因A、D抗原位点在杆状病毒中成功表达,其表达产物为TGE诊断试剂的制备、基因工程疫苗的研制奠定了基础。 相似文献
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应用生物信息学软件对猪传染性胃肠炎病毒(TGEV)S基因进行分析,筛选出可能与S基因有相互作用的外源miRNA:amiRNA-S-28035,amiRNA-S-28038,amiRNA-S-28165。之后,利用脂质体将构建好的相应的表达载体瞬时转染至PK-15细胞;通过Q-PCR和间接免疫荧光方法检测其对S基因的抑制作用;CPE分析和TCID50测定检测其对TGEV增殖的抑制效果。结果发现,3个外源性miRNA均降低了S基因mRNA的转录和蛋白的表达,其中amiRNA-S-28038对TGEV mRNA的平均抑制率可达64.6%,最高可达69.9%,表明外源性microRNA可以通过靶向TGEV基因组来抑制TGEV的复制。研究结果为猪传染性胃肠炎的预防和治疗提供了新的思路。 相似文献
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根据猪传染性胃肠炎病毒(TGEV)纤突(S)蛋白的全基因序列及表达载体质粒的基因融合特点,设计一对引物,进行PCR,获得含有TGEV S基因4个主要抗原位点的约2000 bp目的片段,将其分别与表达载体质粒pPG611.1和pPG612.1进行连接,通过电转化进入宿主菌Lacto-bacillus casei393细胞内,通过质粒提取、PCR鉴定、酶切鉴定和序列测定分析,表明TGEV S基因已成功插入到表达载体质粒中,获得了TGEV S蛋白干酪乳杆菌表达载体系统。 相似文献
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猪传染性胃肠炎病毒分子生物学研究进展 总被引：2，自引：0，他引：2
猪传染性胃肠炎病毒(TGEV)可引起猪传染性胃肠炎,给养猪业造成极大危害。本文介绍TGEV基因组结构、S基因及结构蛋白与功能,通过对TGEV的分子生物学特性的深入研究,对目前开发研制的新型疫苗具有重要意义。 相似文献
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《动物医学进展》2016,(8)
猪传染性胃肠炎(TGE)是严重危害全世界养猪业健康发展的一种高度传染性病毒病,其病原猪传染性胃肠炎病毒(TGEV)的S蛋白携带有主要的B淋巴细胞识别的抗原决定簇,是诱导机体产生保护性中和抗体的主要抗原。本研究以提取的TGEV陕西分离株感染的ST细胞的RNA为模板,采用RTPCR方法扩增编码S蛋白上包含C、B、D、A 4个抗原位点的基因片段,将目的片段连接到真核表达载体pcDNA3.1/V5-HisB上,构建重组表达载体,转染293T细胞后,通过Western blot检测目的基因的表达。结果成功克隆到编码S蛋白C、B、D、A 4个抗原位点的基因片段,以TGEV阳性猪血清和抗His标签单抗的Western blot结果均显示,目的基因在293T细胞中获得表达。本研究为TEG核酸疫苗的研制奠定了基础。 相似文献
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携带猪传染性胃肠炎病毒S/N融合双基因的减毒沙门氏菌的构建与鉴定 总被引：1，自引：0，他引：1
旨在构建携带猪传染性胃肠炎病毒(TGEV)S/N融合双基因的减毒沙门氏菌,并鉴定该疫苗菌株的生物学特性,为开展TGEV口服免疫研究奠定材料基础。采用PCR方法从克隆质粒19T-S和19T-N中分别扩增了TGEV的S基因(含主要抗原位点,2.1kb)和N基因(1.2kb),将S基因和N基因插入pVAX1载体,构建携带S/N融合双基因的真核表达质粒pVAX-S/N。将pVAX-S/N电转化减毒沙门氏菌SL7207,筛选获得重组菌株SL7207(pVAX-S/N),并对重组菌株SL7207(pVAX-S/N)的体外稳定性、目的基因在体内的转录、口服接种小鼠的安全性及在体内稳定性等特性进行了鉴定。结果表明,真核质粒pVAX-S/N构建成功,该质粒转染COS7中能表达2个目的蛋白,重组菌SL7207(pVAX-S/N)在Kan+抗性下体外培养稳定性好,口服接种小鼠3d可从回肠组织检测到目的基因的转录,以0.5×109、1×109和2×109 CFU口服对小鼠均具有安全性,重组菌在接种小鼠的肝、脾于4周左右逐渐被机体清除。结果表明成功构建TGEVS/N双基因疫苗SL7207(pVAX-S/N),该疫苗具有良好的稳定性与安全性等特点,为开展TGEV口服免疫研究奠定了基础。 相似文献
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将RT-PCR扩增的猪传染性胃肠炎病毒（TGEV）S基因A抗原位点目的片段克隆、双酶切后连接于表达载体,经酶切、PCR和测序鉴定的阳性重组质粒转化BL21（DE3）,用IPTG诱导,进行SDS-PAGE和Western-blotting分析,确定目的蛋白的原核表达情况和免疫特异性。结果显示,目的片段的大小为534bp,序列分析表明,该基因与其他TGEV相应基因具有很高的同源性;Western-blotting检测可见分子质量约43 ku的融合蛋白条带,表明,该蛋白具有良好的反应原性。 相似文献
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猪传染性胃肠炎病毒S基因B、C位点的克隆及原核表达载体的构建 总被引：1，自引：0，他引：1
参考GenBank上公布的猪传染性胃肠炎病毒（TGEV）S基因B、C抗原位点序列,应用Primer6．0设计一对含酶切住点的引物,用于RT—PCR扩增B、C抗原位点目的片段,将扩增产物连接于peasy—T克隆载体上,构建克隆载体,用EcoRⅠ和XhoⅠ对表达载体PET-32a（＋）和重组质粒进行酶切,将酶切产物亚克隆至PET-32a（＋）多克隆位点上,连接、转化至BL21（DE3）,构建B、C位点的原核表达载体,并对阳性重组质粒进行酶切、PCR和测序鉴定。所扩增的目的片段的大小为357bp,与原核表达载体连接后,经核苷酸及推导的氨基酸序列分析表明,该基因与其他猪传染性胃肠炎病毒相应基因具有很高的同源性,说明成功地构建了TGEVS基因B、C抗原位点的原核表达载体。TGEVS基因B、C抗原位点原核表达载体的成功构建,为TGEV诊断方法的建立提供良好的技术基础。 相似文献
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魏静 《四川畜牧兽医学院学报》2009,(4):28-32
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。 相似文献
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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。 相似文献
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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. 相似文献
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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。 相似文献
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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. 相似文献