首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献，搜索用时 93 毫秒
1.
为了找到一种获得鸡白细胞介素-18(IL-18)活性蛋白的简便方法,试验采用基因工程的方法,以鸡脾淋巴细胞总RNA为模板进行RT-PCR扩增,克隆鸡IL-18成熟蛋白基因并进行序列分析。将鸡IL-18成熟蛋白基因克隆至原核表达载体pGEX-KG,构建原核表达质粒,通过异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导重组鸡IL-18蛋白在大肠杆菌中的表达,经葡聚糖凝胶层析柱纯化后,用3-(4,5)-双甲基-2-噻唑-(2,5)-二苯基溴化四氮唑蓝(MTT)法检测表达重组蛋白的生物学活性。结果表明:鸡IL-18成熟蛋白基因开放阅读框为510 bp,编码170个氨基酸;所获得的鸡IL-18成熟蛋白基因与GenBank中鸡IL-18的核苷酸同源性为99.0%～99.8%,氨基酸同源性为97.5%～99.5%;成功地构建了pGEX-IL-18原核表达质粒,表达的重组蛋白质量约为45.6 ku,该重组蛋白具有明显增强鸡脾淋巴细胞增殖的生物学活性。  相似文献   

2.
鸡IL-2基因的克隆及GST-chIL2融合蛋白的表达   总被引：1，自引：1，他引：0  
根据Sundick等发表的鸡IL-2基因(chIL2)序列设计合成特异性引物,用RT-PCR从ConA诱导的鸡脾淋巴细胞扩增出450 bp的目的片段,酶切鉴定及序列测定结果表明为鸡IL-2基因。该基因包括鸡IL-2基因的全部开放阅读框,编码142个氨基酸组成的蛋白质,与GenBank鸡IL-2基因相比,在编码氨基酸的49位有一个氨基酸缺失;而与Broiler、SC、Chenren和Xiaoshan鸡在编码氨基酸上完全一致,具有较近的亲缘关系;与Kestrel来航鸡、来航SPF鸡、Obese、Silky和Xianju鸡等有1-4个氨基酸的差异;与火鸡和鹌鹑的氨基酸同源性分别为69.9%和59.4%。将克隆到的基因插入到融合蛋白原核表达载体pGEX-6p-1中,得到重组表达质粒pGEX-IL2。将此重组质粒转化大肠杆菌DH5α,经IPTG诱导,表达出了大小约为40 ku的GST-chIL2融合蛋白,其中GST部分为26 ku,鸡IL-2为14 ku,与预期的鸡IL-2成熟蛋白大小一致。  相似文献   

3.
鸡催乳素和抑制素-α亚基重组蛋白的构建及表达   总被引：3，自引：0，他引：3  
将鸡催乳素 (PRL)和抑制素 - α亚基 (INB- α)基因编码序列重组为融合基因 ,制备了同时包含这 2种激素基因的融合蛋白。通过 PCR和分子克隆的方法首先将全部粤黄鸡 PRL成熟肽 c DNA克隆到载体 p RSET A的 Bgl 和 Eco R 克隆位点之间 ,获得重组质粒 p PRL- RSET。鸡 INB- α片段经扩增后分别被克隆到质粒 p RSET A和 p PRL- RSET的 Nhe 和 Xho 克隆位点之间 ,获得重组质粒 p INB- RSET和 p INB- PRL。以上重组质粒构建的正确性分别由各特定引物组合扩增的 PCR产物长度、特定限制性内切酶消化各重组质粒所得产物长度以及对各质粒的测序结果得到验证。重组质粒 p PRL- RSET和 p INB- PRL 转化 E.coli BL2 1(DE3)株 ,IPTG诱导后所表达的产物经 SDS- PAGE显示 ,其分别与所预期的重组蛋白分子大小相符。质粒 p PRL - RSET和 p INB- PRL的表达产物和用 Ni- NTA凝胶纯化的 2重组蛋白产物都可与抗鸡 PRL 抗体产生特异的免疫印迹 ,并且表达菌裂解液和相应纯化蛋白的免疫印迹处于同一位置。结果说明 ,试验已成功完成了鸡 PRL、INB-α及 2者融合蛋白的构建、表达和纯化  相似文献   

4.
绿脓杆菌外毒素A受体结合区蛋白基因重组与鉴定   总被引：2，自引：1，他引：1  
为了获得高表达量重组绿脓杆菌外毒素 A(PEA)受体结合区蛋白 ,以用作免疫原 ,从含有目的片段 (10 0 0 bp)的 p ET- 2 0 (b) B1 0 质粒切下目的片段 ,以正确阅读框架插入含有 T7lac启动子的 p ET- 2 8c( ) DNA中 ,构建了p ET- 2 8c( ) B1 0 质粒。用该质粒转化大肠杆菌 BL2 1 (DE3 )、BL2 1 (DE3 ) p L y S,经 IPTG诱导 ,BL2 1 (DE3 ) p L y S能高效表达目的蛋白。 SDS- PAGE、Western blot分析证实 ,该蛋白即是所需的蛋白 ,该重组蛋白占菌体总蛋白的 5 8%。  相似文献   

5.
鸡白细胞介素-18基因的原核表达及多克隆抗血清的制备   总被引：9，自引：0，他引：9  
将编码鸡白细胞介素-18(interleukin-18，IL-18)成熟蛋白的基因亚克隆至原核表达载体pPROEXTMHT中，构建重组质粒并进行确证性序列测定。然后将重组质粒转化大肠杆菌DH5α并用IPTG于37℃诱导培养。表达的融合蛋白主要以包涵体形式存在，目的蛋白表达量占菌体蛋白的30％。SDS-PAGE和Western blot分析显示，表达的鸡IL-18融合蛋白相对分子质量约为23000。包涵体被6mol／L盐酸胍裂解后，通过镍离子亲和树脂进行了纯化。用所获得的重组鸡IL-18融合蛋白及其纯化产物经3次肌肉注射豚鼠，制备豚鼠抗鸡IL-18多克隆抗血清，并用琼脂扩散试验多克隆抗血清进行了证明。本试验成功的原核表达、纯化了鸡IL-18融合蛋白，并制备了抗鸡IL-18多克隆抗血清，为下一阶段关于鸡IL-18重组蛋白生物学特性及其应用的研究打下了坚实的基础。  相似文献   

6.
将 J亚群禽白血病病毒 (AL V- J)急性转化型中国分离毒 SD990 2株的 env- gp85基因的 PCR产物克隆进表达性载体 p GEX- 5 X- 3中 ,构建成重组质粒 p GEX- SD990 2 - gp85。将重组质粒 p GEX- SD990 2 - gp85转化的 BL 2 1大肠杆菌培养并用 IPTG诱导后 ,其菌体的超声波裂解物用 SDS- PAGE电泳分离蛋白质 ,在经考马斯亮蓝染色的 SDS- PAGE凝胶中与非重组载体 p GEX- 5 X- 3的转化菌裂解物相比 ,重组质粒 p GEX- SD990 2 - gp85的转化菌失去了相对分子质量为2 90 0 0的天然谷胱甘肽 (GST)蛋白带 ,但出现 1条相对分子质量为 6 30 0 0的条带 (相当于 AL V- J囊膜 gp85蛋白与GST的融合蛋白的相对分子质量 )。在 Western- blot中 ,AL V- J特异性单抗 JE9仅能从重组质粒 p GEX- SD990 2 - gp85的转化菌中特异性地识别出该 6 30 0 0的蛋白条带 ,而与天然非重组载体 p GEX- 5 X- 3的转化菌不发生反应。而抗 GST的抗体却从天然非重组载体 p GEX- 5 X- 3和重组质粒 p GEX- SD990 2 - gp85的转化菌中分别识别出 2 90 0 0或 6 30 0 0的条带。 Western- blot的结果证明 ,能分别被 AL V- J特异性单抗 JE9及 GST特异性抗体识别的相对分子质量为 6 30 0 0的蛋白质 ,确实为 GST与 AL V- Jgp85蛋白的融合蛋白  相似文献   

7.
  相似文献   

8.
空肠弯曲菌共同抗原基因文库的建立与基因克隆   总被引：1，自引：0，他引：1  
  相似文献   

9.
将克隆得到的鸡 IFN- γ基因亚克隆到大肠杆菌原核表达载体 p PROEXTMHT的 Pst 和 Kpn 位点上 ,构建了重组表达质粒 p PROEXTMHT- IFN -γ,经序列分析鉴定 ,确证目的基因克隆入载体的预期位点。将重组质粒转化大肠杆菌 DH5 α,于 37℃不同时间诱导培养 ,SDS- PAGE电泳表明 ,该基因在大肠杆菌中发生高水平表达 ,表达的鸡 IFN- γ融合蛋白相对分子质量约为 2 2 0 0 0。重组蛋白占菌体总蛋白的 33%。经 Western blot鉴定 ,该重组蛋白质具有生物学活性。包涵体被 6 mol/ L 盐酸胍裂解后 ,通过镍离子亲和树脂进行了纯化。用所获得的重组鸡 IFN- γ融合蛋白及其纯化产物经 3次肌肉注射于新西兰白兔 ,制备了兔抗鸡 IFN- γ多克隆抗体。琼脂扩散结果表明 ,多克隆抗体可与纯化的鸡IFN -γ重组蛋白发生特异性反应  相似文献   

10.
采用 RT- PCR方法从猪脂肪组织扩增出瘦蛋白基因 ,将其插入 p MD1 8- T克隆载体 ,经序列分析证明 ,所获得的目的基因为 4 4 1 bp,与预期大小一致。提取质粒后用 Eco R 和 Xho 双酶切 ,克隆入 p ET- 2 8a表达载体 ,将阳性重组质粒转化表达受体菌 E.coli BL 2 1 ( DE3) ,经 IPTG诱导 ,SDS- PAGE检测 ,证明在 E.coli BL 2 1中正确表达了重组猪瘦蛋白  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.
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   

14.
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   

15.
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   

16.
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   

17.
The occurrence of ectoparasites in sheep flocks is frequently reported but seldom quantified. Sheep production used to be a predominantly family activity in the state of Sa~o Paulo (Brazil), but it began to become a commercial activity in the past decade. Thus, information about the ectoparasites existing in sheep flocks has become necessary. The present data were obtained by means of questionnaires sent to all sheep breeders belonging to the `Associaça~o Paulista de Criadores de Ovinos' (ASPACO; Sa~o Paulo State Association of Sheep Breeders). Response reliability was tested by means of random visits paid to 10.6% of the respondents. Most of the properties (89.5%) reported the presence of one or more ectoparasites. Screw-worm (Cochliomyia hominivorax) was the most frequent ectoparasite (72.5%), followed by bot fly larvae (Dermatobia hominis, 45.0%), ticks (Amblyomma cajennense) and Boophilus microplus, 31.3%) and finally lice (Damalinia ovis, 13.8%). Combined infestations also occurred, the most common one being screw-worm with bot fly larvae (36.0%) followed by bot fly larvae with ticks (13.9%), screw-worm with ticks (9.3%), bot fly larvae with lice (6.9%), and ticks with lice (5.0%). The most common triple combination was screw-worm, bot fly larvae and ticks (12.8%). Breeds raised for meat or wool were attacked by bot fly larvae and ticks more often than other breeds. Lice were only absent from animals of indigenous breeds. The relationships among these ectoparasites are discussed in terms of sheep breeds, flock size, seasonality and the ectoparasitic combinations on the host.  相似文献   

18.
The purpose of this study was to determine the frequency of different tumor types within a large cohort of cats with intracranial neoplasia and to attempt to correlate signalment, tumor size and location, and survival time for each tumor. Medical records of 160 cats with confirmed intracranial neoplasia evaluated between 1985 and 2001 were reviewed. Parameters evaluated included age, sex, breed, FeLV/FIV status, clinical signs, duration of signs, number of tumors, tumor location(s), imaging results, treatment, survival times, and histopathologic diagnosis. Most of the cats were older (11.3 +/- 3.8 years). Primary tumors accounted for 70.6% of cases. Metastasis and direct extension of secondary tumors accounted for only 5.6 and 3.8% of cases, respectively. Twelve cats (7.5%) had 2 or more discrete tumors of the same type, whereas 16 cats (10.0%) had 2 different types of intracranial tumors. The most common tumor types were meningioma (n = 93, 58.1%), lymphoma (n = 23, 14.4%), pituitary tumors (n = 14, 8.8%), and gliomas (n = 12, 7.5%). The most common neurological signs were altered consciousness (n = 42, 26.2%), circling (n = 36, 22.5%), and seizures (n = 36, 22.5%). Cats without specific neurological signs were common (n = 34, 21.2%). The tumor was considered an incidental finding in 30 (18.8%) cats. In addition to expected relationships (eg, meninges and meningioma, pituitary and pituitary tumors), we found that lesion location was predictive of tumor type with diffuse cerebral or brainstem involvement predictive of lymphoma and third ventricle involvement predictive of meningioma.  相似文献   

19.
Comparison of different tests to diagnose feline infectious peritonitis   总被引：1，自引：0，他引：1  
  相似文献   

20.
An experiment was conducted to study disappearance of C14 to C18 fatty acids, lag times and biohydrogenation (BH) rates of C18 fatty acids of ground soya bean and canola seeds in situ. Three ruminally fistulated Dallagh sheep were used to determine ruminal BH of unsaturated fatty acids (UFAs). Differences in the disappearance of fatty acids through the bags and lag times were observed between the oilseeds. We saw that the longer the incubation time of the oilseeds in the rumen, the lower the content of C18:2 and C18:3. Significantly higher lag times for both C18:2 and C18:3 were observed in ground canola compared to ground soya bean. BH rates of C18:2 and C18:3 fatty acids in soya bean were three times higher than those of canola. These results suggest that the fatty acid profile of fat source can affect the BH of UFAs by rumen micro‐organisms. So that UFAs of canola had higher ability to escape from ruminal BH. It seems that fatty acid profile of ruminant products is more affected by canola seed compared to soya bean seed.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号