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1.
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   

2.
重组菌株BL21(DE3)(pXETSLT1)经IPTG诱导后,其表达产物经SDS-PAGE和ELISA检测,结果表明重组菌株可以高效表达大肠杆菌肠毒素ST1-LTB 融合蛋白,该融合蛋白占菌体总蛋白的33.21% ,而且已失去天然ST1肠毒素生物毒性。用从IPTG 诱导的工程菌中提取的包涵体或经甲醛灭活的工程菌制成抗原免疫小鼠,结果免疫小鼠至少能抵抗1.5MLD 的大肠杆菌强毒株C83902(K88ac,ST+ ,LT+ )的攻击。用提取的包涵体免疫家兔后,采集的血清能够中和天然ST1肠毒素的毒性。这表明构建的工程菌株BL21(DE3)(pXETSLT1)可以作为预防幼畜大肠杆菌性腹泻基因工程菌苗的候选株  相似文献   

3.
大肠杆菌肠毒素ST1-LTB融合基因的高效表达   总被引:13,自引:0,他引:13  
用限制性核酸内切酶EcoRⅠ和HindⅢ双酶切含大肠杆菌肠毒素ST1—LTB融合基因的质粒pXSLT1,回收084kb的ST1—LTB融合基因,再将载体pET—28b(+)用EcoRⅠ和HindⅢ双酶切,然后将其与ST1—LTB融合基因连接,转化至受体菌BL21(DE3)中。经EcoRⅠ、HindⅢ、BamHⅠ酶切反应鉴定重组子质粒,得到了理想重组质粒pXETSLT1。重组菌株BL21(DE3)(pXETSLT1)经IPTG诱导后,其表达产物经SDS—PAGE和ELISA检测,结果表明重组菌株可以高效表达ST1—LTB融合蛋白,该融合蛋白占菌体总蛋白的3321%,而且无天然ST1生物毒性。  相似文献   

4.
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   

5.
大肠杆菌肠毒素ST1—LTB融合蛋白工程菌株的免疫原性研究   总被引:9,自引:1,他引:8  
已构建的能表达大肠杆菌肠毒素ST1LTB融合蛋白的工程菌株BL21(DE3)(pXETSLT1)及其表达产物经动物试验证实没有毒性反应。用从IPTG诱导的工程菌中提取的包涵体或经甲醛灭活的工程菌制成抗原,免疫小鼠,结果免疫小鼠至少能抵抗15MLD的大肠杆菌强毒株C83902(K88ac,ST+,LT+)的攻击。用提取的包涵体免疫家兔后,采集的血清能够中和天然ST1的毒性。这表明构建的工程菌株BL21(DE3)(pXETSLT1)可以作为预防幼畜大肠杆菌性腹泻基因工程菌苗的候选株。  相似文献   

6.
猪生殖—呼吸道综合征病毒CH—1a株N基因的原核表达   总被引:3,自引:0,他引:3  
将PRRSVCH-1a株N基因用EcoRI和PstI双酶切从重组质粒pUC18-ORF7切下后,插入到原核表达载体pBV220的PR,PL启动子下游,得到重组表达质粒,pBV220-ORF7,转化了pBV220-ORF7的大肠杆菌JM83经诱导培养后,用SDS-PAGE和Westernblot检测表达产物,结果表明PRRSVCH-1a株的N基因在原核载体上得到高效表达,表达产物占菌体总蛋白的15.  相似文献   

7.
将PRRSVCH1a株N基因用EcoRI和PstI双酶切从重组质粒pUC18ORF7切下后,插入到原核表达载体pBV220的PR、PL启动子下游,得到重组表达质粒pBV220ORF7。转化了pBV220ORF7的大肠杆菌JM83经诱导培养后,用SDSPAGE和Westernblot检测表达产物。结果表明PRRSVCH1a株的N基因在原核载体上得到高效表达,表达产物占菌体总蛋白的153%。表达蛋白可望成为有价值的PRRS诊断抗原。  相似文献   

8.
含绿脓杆菌外毒素A受体结合区重组蛋白的纯化   总被引:5,自引:3,他引:2  
将表达含绿脓杆菌外毒素(PEA)受体结合区基因的质粒pET-EAB转化到大肠杆菌BL21(DE3)中,经IPTG诱导表达了含PEA受体结合区的重组蛋白(称PE34)。PE34主要以包涵体形式存在于大肠杆菌中,用溶菌酶-脱氧胆酸钠法结合超声波裂解法破碎细菌,离心制备包涵体。用2mol/L脲洗涤包涵体后,包涵体纯度可达75%,在8mol/L脲存在条件下,SephacrylS-200凝胶滤过,DEAE-SepharoseFastFlow离子交换层析纯化,获得SDS-PAGE1条带的PE34,纯度达95.8%,得率为24.5%。  相似文献   

9.
对含有产气荚膜梭菌(Clostridiumperfringens)α毒素基因的重组菌株BL21(DE3)(pXETA1)和BL21(DE3)plysS(pXETA1),通过培养性状观察和小鼠接种试验,证明这2株重组菌株均无致病性。随后对这2株重组菌株的表达产物进行了研究,经SDS-PAGE和薄层凝胶扫描分析,IPTG诱导3~4h后的BL21(DE3)(pXETA1)表达的α毒素占菌体总蛋白33.21%,BL21(DE3)plysS(pXETA1)表达的α毒素占菌体总蛋白27.25%,其相对分子质量约37500;经Westernblot分析,表达产物可被α毒素抗血清识别。包涵体粗提物的免疫攻毒试验结果表明,以1倍致死量攻击的免疫小鼠可获得100%(36/36)的保护,以2倍致死量攻击的免疫小鼠可获得94.28%(33/35)的保护,从而说明表达产物具有良好的免疫原性。  相似文献   

10.
HIV—2env基因在大肠杆菌和重组痘苗病毒中的表达   总被引:1,自引:0,他引:1  
将编码人Ⅱ型免疫缺陷病毒(HIV-2)envgp120(E1)和gp36(E2)分别克隆到原核高效表达载体pET17b、pBV220和真核表达载体p10、p16中,构建成8个重组质粒。经SDS-PAGE和Westernblot分析证明,在原核表达系统成功地表达了HIV-2env融合蛋白,表达的蛋白分子量分别为35000、70000和50000,而原核表达质粒pETE1在SDS-PAGE凝胶的35000处可见明显的额外蛋白带。经薄层扫描测定,原核系统表达的Env蛋白(gp120)相对含量为5.8%。在真核细胞中表达的Env蛋白经Westernblot检测分析,分子量分别为60000、57000和42000。上述表达的Env蛋白均能与HIV-2阳性血清发生特异性反应。重组痘苗病毒可诱导小鼠产生抗HIV-2Env抗体  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.
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   

17.
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   

18.
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   

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.
The epidemiology of H. placei and of other gastrointestinal nematodes in yearling dairy cattle was examined on two farms in Kiambu District, central Kenya during each of 13 one-month periods from April 1993 to April 1994. On each farm, 32 newly weaned dairy calves were given a single dose of albendazole and then placed on experimental pastures. Twelve of the animals were designated for bi-monthly slaughter (n = 2) and analysis of worm population characteristics and 20 were designated for blood and faecal collection and for weighing. Two parasite-free tracer calves were grazed alongside the weaner calves each month throughout the study period and were also slaughtered for analysis of worm populations. Faecal egg counts, haematological and serum pepsinogen determinations, herbage larval counts, and animal live weight changes were recorded monthly. The study revealed that Haemonchus placei, Trichostrongylus axei, Cooperia spp. and Oesophagostomum radiatum were responsible for parasitic gastroenteritis and that H. placei was the predominant nematode present in the young cattle on both farms. Faecal egg counts from resident cattle and necropsy worm counts revealed that pasture larval levels were directly related to the amount of rainfall. The total worm burdens in the animals were highest during the rainy season (March–June and October–December) and lowest during the dry seasons (July–September and January–February). The very low recovery of immature larvae of H. placei from the tracer calves indicated that arrested development is not a feature of the life cycle of this parasite in central Kenya. The maintenance of the parasite population depended on continuous cycling of infection between the host and the pasture. The agroclimatic conditions of the study area were such that, in general, favourable weather conditions for the development and survival of the free-living stages of gastrointestinal nematodes existed all year round.  相似文献   

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