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1.
根据已报告的传染性法氏囊病病毒( I B D V) c D N A 序列,在病毒 V P2 区域,设计 1 对引物,并在 2 引物上分别加 Pst Ⅰ和 Eco R I酶切位点。对 2 个 I B D V 分离株 J S3 和 J S4,用异硫氰酸胍酚氯仿抽提一步法提取病毒 R N A,然后进行逆转录和 P C R 扩增,将扩增片段经酶切纯化后克隆于 p U C18 质粒载体中,以双脱氧链末端终止法测定 c D N A 序列。将测定的分离株与标准对照株( S1) I B D V V P2 基因片段的核苷酸序列进行计算机分析比较, J S3 与 S1 的同源性为 97% , J S4 与 S1 的同源性为 97% , J S3 与 J S4 的同源性为 99% 。推导编码蛋白氨基酸序列, J S3 和 S1 的同源性为 97% , J S4 与 S1 的同源性为 96% , J S3 和 J S4 的同源性为 98% 。这表明在我国流行的 I B D V 毒株与标准毒株相比不仅在免疫学反应上有差异,而且在病毒核酸序列上有变异,这种变异是造成疫苗免疫失败或免疫逃避的分子基础。  相似文献   

2.
利用反转录套式聚合酶链反应技术从北京地区3株鸡性支气管炎病毒(IBV)分离株中扩增出1054bpS1基因高变区。利用限制性内切酶SinI和PstⅠ的酶切位点分析图谱进一步证实了RT-nestedPCR的特异性。将3段S1基因分别克隆到PUC19质粒中,获得重组质粒PUCIBVS1-BJ1,PUCIBVS1-BJ2和PUCIBVS1-BJ3。  相似文献   

3.
狂犬病病毒糖蛋白基因在原核细胞中的表达   总被引:2,自引:0,他引:2  
将狂犬病病毒糖蛋白(RVgp)基因BglⅡ片段(1675bp)分别正向插入到原核高效表达载体pET-17b和pET-17b2(用SacⅠ-NdeⅠ缺失掉pET-17b60bp含起始密码子ATG小片段)的BamHⅠ切点,构建重组质粒pET-17bRVgp和pET-17b2RVgp。将其分别转化表达受体菌E.coliBL21(DE3)和E,coliBL21(DE3)plysS.IPTG诱导表达,菌体经超声波裂解处理后SDS-pAGE,染色,在分子量约60000处可见重组质粒表达的较宽的蛋白带,以抗RVgpMcAb进行Western-blot检测,表明该表达蛋白为RVgp。通过扫描显示,表达的RVgp占菌体总蛋白的10%~14%,其中pET-17b2RVgp在E。coliBL21(DE3)中的表达量最高。  相似文献   

4.
从母牛接种过A组BRV株NCDV-Lincoln(P1:G6)苗的两个吡邻牛场分离到两株牛轮状病毒(BRV),编号为NS-1和NS-2,Northern-blotting试验表明,NS-1和NS-2有相的的A组RNA电泳模式,而且全部基因片段同源,体外中和试验表明,NS-1株病毒能被G6特异性单克隆抗体(McAs)和抗BRVB641株(P5:G6)豚鼠高免血清(GPAS)中和,但不被G10特异性M  相似文献   

5.
类志贺氏菌毒素Ⅱ型变异体B亚单位基因的表达与鉴定   总被引:8,自引:0,他引:8  
将由 P C R 扩增并克隆在p U C18 质粒载体中的sltⅡe B基因切出,并按预定的阅读框架插入表达性质粒载体 p G E X6p1 中的谷胱苷肽转移酶( G S T)基因的下游。重组质粒 pppsltⅡe B在大肠杆菌 B L21 中以融合蛋白的形式表达 S L TⅡe B蛋白(命名为 G S T S L TⅡe B),即 S L TⅡe B蛋白(7568k D)与谷胱苷肽转移酶(27335k D)相连组成分子量为 34885k D 的融合蛋白。通过对重组大肠杆菌 P P S L TⅡe B的菌体裂解物的 S D S P A G E 电泳分析,以及根据抗原抗体结合反应的免疫学特性,通过 Westernblot 鉴定,重组大肠杆菌 P P S L TⅡe B(含有重组质粒 ppsltⅡe B的大肠杆菌 B L21)可以大量表达融合蛋白形式的 S L TⅡe B。根据 G S T 可与其底物谷胱苷肽结合的特性,用谷胱苷肽结合的 Sepharose 4 B制备的亲和凝胶纯化表达产物,纯化的表达产物经 S D S P A G E,可以鉴定到分子量约为 35k D 的蛋白条带,这与融合蛋白形式的 S L TⅡe B分子量(34885k D)相当。  相似文献   

6.
伪狂犬病病毒鄂A株TK基因的克隆及其鉴定   总被引:6,自引:0,他引:6  
合成了 1 对能对伪狂犬病病毒( Pseudorabies virus, P R V) T K(thym idine kinase)基因+ 119~+ 1 071区进行特异扩增的引物,用猪 P R V 鄂 A 株细胞培养物提取的基因组作模板,扩增出 953 bp 长的片段,用地高辛标记该片段作探针,通过 South ern 杂交,从克隆有 P R V 鄂 A 株的 Bam H I片段的重组质粒中钓出含 T K 基因的重组质粒 p S T K。对 p S T K 进行酶切分析,绘制了含 T K 基因的 Bam H I片段的图谱,通过测序得出了 T K 基因的全序列。将该序列与 P R V N I A3 株 T K 基因进行比较,发现鄂 A 株的 T K 基因存在变异。  相似文献   

7.
传染性支气管炎病毒中国地方分离株RFLP基因分型的研究   总被引:5,自引:1,他引:4  
为初步确定我国不同地区流行的传染性支气管炎病毒(IBV)的基因分型,对分离自国内8个不同地域疫区的IBVQD、GZ、ZZ、TJ、DL、YC、JS1和JS2及参考株M41、H52和T的S1基因RT-PCR扩增cDNA进行HaeⅢ的RFLP分析。结果,QD与MD41、H52同属Massachussete基因型,GZ、ZZ、YC与T的基因型相同,DL、JS1和JS2则表现为各自独立的基因型,而TJ则为DL和T2种基因型毒株的混合感染,表明我国的广大地域内存在着Mass基因型、T基因型和可能的变异株IBV的流行。  相似文献   

8.
从中国发病鸡群中分离的鸡减蛋综合征病毒(EDSV)AA-2株,经常规方法提取其病毒核酸后,构建了限制性内切酶PstⅠ及HindⅢ水解片段的基因文库。对其中HindⅢ-F片段(52.9~58.9mu)的正反2条链的序列测定发现,其反链存在1个编码容量为387个氨基酸(aa)的开放读码框架(openreadingframe,ORF),经Genebank/EMBL同源搜寻后证实其编码产物为EDSV的DNA结合蛋白(DBP)。与其他腺病毒DBP的氨基酸序列进行同源比较,其N端同源性在19.0%~46.2%之间,C端同源性在27.7%~60.4%之间。腺病毒DBP的3个保守序列CR1,CR2,CR3在EDSVDBP中有较大变化,但EDSVDBP的锌指框架(Zn2+fingermotif)却保留了对其功能必需的残基。  相似文献   

9.
传染性法氏囊病病毒VP2基因的克隆和鉴定   总被引:1,自引:0,他引:1  
以鸡胚成纤维细胞培养IBDV弱毒疫苗株D78,经蔗糖密度梯度离心纯化,电镜下见典型IBDV粒子。用SDS-蛋白酶K法提取病毒基因组,低熔点胶回收,琼脂糖凝胶电泳,可见IBDV典型双节段基因组。根据已发表的IBDV基因组序列,设计并合成了1对特异扩增IBDVVP2基因的引物。以IBDV基因组为模板,利用RT-PCR技术扩增出了1.5kb的cDNA产物,将PCR产物适当处理后与经SmaⅠ酶切的pUC19质粒平端连接,转化大肠杆菌DH5α,在含Amp、Χ-gal和IPTG的琼脂平板上培养,产生大量白色菌落。挑取白色菌落,经质粒大小比较分析初步筛选到一重组质粒。以重组质粒DNA为模板作PCR检测和部分酶切分析证实,该质粒为含D78IBDVVP2基因的重组pUC19  相似文献   

10.
弓形虫主要表面抗原P30基因克隆与表达   总被引:2,自引:0,他引:2  
将液氮保存的弓形虫 N T 株经小鼠复壮后,取腹腔液提取弓形虫基因组 D N A,采用 P C R 方法从弓形虫 N T 株中扩增出约 800 bp 的片段。产物经 Eco R I和 Xba I酶切后,克隆到 p U C19 载体中,构建了 p B V P30 非融合表达质粒和 p E T P30 融合表达质粒。p B V P30 转化到宿主菌 D H5α、p E T P30 转化到宿主菌 B L21 ( D E3)后,分别经温控诱导和 I P T G 诱导,产物经 S D S P A G E 分析,p B V P30 未发现表达产物,p E T P30 出现约 30 000 的产物。 W estern blotting 显示,该蛋白与兔抗弓形虫血清发生特异性反应;薄层扫描显示,该蛋白占菌体总蛋白的 20% 以上。  相似文献   

11.
试验选用96头平均体重14.82 kg左右的杜×长×大断奶仔猪,随机分成4组,每组3栏,每栏8头(公母各半)。对照组饲喂基础日粮,试验1、2、3组分别添加1%80目白术、0.2%白术多糖和1%微米白术。试验期30 d。结果表明:在生长性能方面,与对照组相比,1%微米白术添加组可显著提高日增重(P0.05)、降低饲料增重比和腹泻率,而且效果优于1%80目白术组和0.2%白术多糖组,在肠道形态和肠道微生态区系方面,与对照组相比,日粮添加1%80目白术、0.2%白术多糖、1%微米白术均可不同程度的提高十二指肠和空肠的绒毛高度,加深十二指肠和空肠的隐窝深度,并且增加肠道微生态区系的多样性,其中以1%微米白术添加组的效果最佳。  相似文献   

12.
Sissay, M.M., Uggla, A. and Waller, P.J., XXXX. Prevalence and seasonal incidence of nematode parasites and fluke infections of sheep and goats in eastern Ethiopia. Tropical Animal Health and Production, XXXX. A 2-year abattoir survey was carried out to determine the prevalence, abundance and seasonal incidence of gastro-intestinal (GI) nematodes and trematodes (flukes) of sheep and goats in the semi-arid zone of eastern Ethiopia. During May 2003 to April 2005, viscera including liver, lungs and GI tracts were collected from 655 sheep and 632 goats slaughtered at 4 abattoirs located in the towns of Haramaya, Harar, Dire Dawa and Jijiga in eastern Ethiopia. All animals were raised in the farming areas located within the community boundaries for each town. Collected materials were transported within 24 h to the parasitology laboratory of Haramaya University for immediate processing. Thirteen species belonging to 9 genera of GI nematodes (Haemonchus contortus, Trichostrongylus axei, T. colubriformis, T. vitrinus, Nematodirus filicollis, N. spathiger, Oesophagostomum columbianum, O. venulosum, Strongyloides papillosus, Bunostomum trigonocephalum, Trichuris ovis, Cooperia curticei and Chabertia ovina), and 4 species belonging to 3 genera of trematodes (Fasciola hepatica, F. gigantica, Paramphistomum {Calicohoron} microbothrium and Dicrocoelium dendriticum) were recorded in both sheep and goats. All animals in this investigation were infected with multiple species to varying degrees. The mean burdens of adult nematodes were generally moderate in both sheep and goats and showed patterns of seasonal abundance that corresponded with the bi-modal annual rainfall pattern, with highest burdens around the middle of the rainy season. In both sheep and goats there were significant differences in the mean worm burdens and abundance of the different nematode species between the four geographic locations, with worm burdens in the Haramaya and Harar areas greater than those observed in the Dire Dawa and Jijiga locations. Similar seasonal variations were also observed in the prevalence of flukes. But there were no significant differences in the prevalence of each fluke species between the four locations. Overall, the results showed that Haemonchus, Trichostrongylus, Nematodirus, Oesophagostomum, Fasciola and Paramphistomum species were the most abundant helminth parasites of sheep and goats in eastern Ethiopia.  相似文献   

13.
14.
1-(2-Chloroethyl)3-cyclohexyl-1-nitrosourea (CCNU) is an alkylating agent in the nitrosourea subclass. A prospective evaluation of CCNU was done to determine the maximally tolerated dosage of CCNU in tumor-bearing cats. Response data were obtained when available. Twenty-five cats were treated with CCNU at a dosage of 50-60 mg/m3 body surface area. Complete hematologic data were available for 13 cats. Neutropenia was the acute dose-limiting toxicity. The median neutrophil count at the nadir was 1,000 cells/microL (mean, 2,433 cells/microL; range, 0-9,694 cells/microL). The time of neutrophil nadir was variable, occurring 7-28 days after treatment, and counts sometimes did not return to normal for up to 14 days after the nadir. Based on these findings, a 6-week dosing interval and weekly hematologic monitoring after the 1st treatment with CCNU are recommended. The nadir of the platelet count may occur 14-21 days after treatment. The median platelet count at the nadir was 43,500 cells/microL. No gastrointestinal, renal, or hepatic toxicities were observed after a single CCNU treatment, and additional studies to evaluate the potential for cumulative toxicity should be performed. Five cats with lymphoma and 1 cat with mast cell tumor had measurable responses to CCNU. Phase II studies to evaluate antitumor activity should be completed with a dosing regimen of 50-60 mg/m3 every 6 weeks.  相似文献   

15.
Our particular attention in this article was given to natural mediators for macrophages isolated from the sites of tissue injury. A number of chemotactic factors, which may satisfy many criteria making them acceptable as inflammatory leucocyte chemotactic factors, has been separated. Among them, our laboratory has isolated three macrophage (monocyte) chemotactic factors (MCF-a, -b and -c). Their purification, characterization and functional specificity are discussed.  相似文献   

16.
Genetic variations in chromosome Y are enabling researchers to identify paternal lineages, which are informative for introgressions and migrations. In this study, the male‐specific region markers, sex‐determining region‐Y (SRY), amelogenin (AMELY) and zinc finger (ZFY) were analysed in seven Turkish native goat breeds, Angora, Kilis, Hair, Honaml?, Norduz, Gürcü and Abaza. A SNP in the ZFY gene defined a new haplotype Y2C. All domestic haplogroups originate from Capra aegagrus, while the finding of Y1A, Y1B, Y2A and Y2C in 32, 4, 126 and 2 Turkish domestic goats, respectively, appears to indicate a predomestic origin of the major haplotypes. The occurrence of four haplotypes in the Hair goat and, in contrast, a frequency of 96% of Y1A in the Kilis breed illustrate that Y‐chromosomal variants have a more breed‐dependent distribution than mitochondrial or autosomal DNA. This probably reflects male founder effects, but a role in adaptation cannot be excluded.  相似文献   

17.
Human and equine athletes are reported to have a high prevalence of gastric disease, and anecdotal evidence suggests a similar phenomenon applies to racing sled dogs. To investigate the prevalence of gastric disease in racing sled dogs, we conducted 2 gastroscopy studies on dogs competing in the annual Iditarod Sled Dog Race. A pilot study of dogs that were either dropped from the 2000 Iditarod Sled Dog Race because of illness or that finished the race indicated that, approximately 5 days after competing, 10 of 28 dogs (35%) had endoscopic evidence of gastric ulceration, erosion, or hemorrhage. The next year, an endoscopic study of 73 dogs participating in the 2001 Iditarod race was performed in order to evaluate a larger population of dogs. Data from 70 of these dogs could be used; 34 (48.5%) had ulceration, erosion, gastric hemorrhage, or some combination of these findings. When this group of 70 dogs was compared retrospectively to a control group of 87 dogs presented to the Texas A&M University (TAMU) Veterinary Medical Teaching Hospital, the Iditarod sled dogs had a significantly higher prevalence (P = .049) of gastric lesions. These findings suggest that, similar to athletes of other species, elite canine athletes have an increased prevalence of gastric disease compared to the canine population at large.  相似文献   

18.
Continuous rate infusion (CRI) of furosemide in humans is considered superior to intermittent administration (IA). This study examined whether furosemide CRI, compared with IA, would increase diuretic efficacy with decreased fluid and electrolyte fluctuations and activation of the renin-angiotensin-aldosterone system (RAAS) in the horse. Five mares were used in a crossover-design study. During a 24-hour period, each horse received a total of 3 mg/kg furosemide by either CRI (0.12 mg/kg/h preceded by a loading dose of 0.12 mg/kg IV) or IA (1 mg/kg IV q8h). There was not a statistically significant difference in urine volume over 24 hours between methods; however, urine volume was significantly greater after CRI compared with IA during the first 8 hours ([median 25th percentile, 75th percentile]: 9.6 L [8.9, 14.4] for CRI versus 5.9 L [5.3, 6.0] for IA). CRI produced a more uniform urine flow, decreased fluctuations in plasma volume, and suppressed renal concentrating ability throughout the infusion period. Potassium, Ca, and Cl excretion was greater during CRI than IA (1,133 mmol [1.110, 1,229] versus 764 mmol [709, 904], 102.7 mmol [96.0, 117.2] versus 73.3 mmol [65.0, 73.5], and 1,776 mmol [1,657, 2.378] versus 1,596 mmol [1,457, 1,767], respectively). Elimination half-lives of furosemide were 1.35 and 0.47 hours for CRI and IA, respectively. The area under the excretion rate curve was 1,285.7 and 184.2 mL x mg/mL for CRI and IA, respectively. Furosemide CRI (0.12 mg/kg/h) for 8 hours, preceded by a loading dose (0.12 mg/kg), is recommended when profound diuresis is needed acutely in horses.  相似文献   

19.
The objective of this report was to characterize 20-year changes in proportion of calcium oxalate (CaOx) calculi and struvite calculi in dogs, and associations with breed, age, and sex. In this retrospective study, results of analysis of urinary calculi from dogs were reviewed for specimens received between July 1, 1981, and December 31, 2001. Breed, sex, age, year of submission of the specimen, and mineral type(s) were analyzed statistically. CaOx or Struvite or both were contained in 18,966 of 20,884 (91%) specimens. For both sexes, a 20-year statistically significant increase was observed in the proportion of calculus specimens that contained CaOx. The increase in this proportion was greater in females (1% to 31%) than in males (18% to 82%). From 1998 to 2001, when proportions may have plateaued, the odds of specimens containing CaOx were markedly higher in 18 breeds, markedly lower in 5 breeds, and not significantly different in 13 breeds compared with crossbreds. For both sexes, a 20-year statistically significant decrease was observed in the proportion of calculus specimens that contained struvite. This decrease in proportion was greater for males (79-16%) than for females (97-68%). From 1998 to 2001, when proportions plateaued, the odds of calculi containing struvite were markedly lower in 20 breeds, markedly higher in 1 breed, and not significantly different in 15 breeds when compared with crossbreds. Breed, age, and sex were associated statistically with CaOx or struvite urolithiasis. In conclusion, there appears to have been a long-term increase in the proportion of specimens of canine urinary calculi that contain CaOx as well as a long-term decrease in the proportion of specimens of calculi that contain struvite for both male and female dogs. The rate of change appeared to begin leveling off in the period 1998 to 2001. The recent proportion of dogs with either CaOx- or struvite-associated urolithiasis may depend on breed, age, and sex, and on interactions among these 3 factors.  相似文献   

20.
<正>1.INTRODUCTION Ensuring transportation network security is one Of the most daunting challenges confronting homeland security agencies today.Significant research has been dedicated.To model and analyze the vulnerability of transportation systems,while notably fewer studies propose specific strategies for deploying defensive technologies to safeguard these systems.  相似文献   

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