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猪蓝耳病病毒抗体双抗原夹心ELISA方法的建立及初步应用 总被引：2，自引：0，他引：2
根据PRRSV VR-2332株序列设计了1对扩增PRRSV N蛋白基因的特异性引物,从PRRSV北方株感染细胞中提取总RNA,通过RT-PCR获得长约372 bp的N蛋白编码基因片段。将其克隆到pGEX-6p-1质粒,构建了原核表达载体pPRRS-N。重组基因在大肠杆菌中表达出相对分子质量约为41 000的融合蛋白,目的蛋白表达量约占菌体蛋白的28.5%。利用此重组融合N蛋白建立了一种检测PRRSV特异性抗体的双抗原夹心ELISA,并通过与商品化试剂盒的应用比较对本方法进行了系统评价。分析了来自北京、山东、河南、河北4省区13个养猪场的260份血清,结果表明,本方法的敏感性和特异性分别为93.5%和86.7%,与IDEXX PRRSV抗体检测试剂盒检测结果的符合率达到91.5%。 相似文献
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从已构建的PRRSV ORF7重组质粒pUCm-T-ORF7中用PCR扩增ORF7基因亚克隆至表达载体pGEX-4T-3,构建重组表达质粒pGEX-4T-3-ORF7并转化大肠杆菌.经SDS-PAGE及Western blotting鉴定,成功表达了谷胱苷肽转移酶(GST)融合的核衣壳蛋白(N),重组N蛋白表达量约为菌体总蛋白的35%,主要以可溶的形式存在,且能形成同源二聚体.重组N蛋白经谷胱苷肽凝胶(glutathione sepharose 4B)亲和层析后得到高度纯化,并将该蛋白作为抗原建立了间接ELISA检测方法.利用该方法对某猪场76份猪血清进行检测并将结果与IDEXX公司ELISA试剂盒检测结果作比较,2种方法的总符合率达93.4%,检测结果之间差异不显著(P＞0.05).结果表明大肠杆菌表达的重组GST融合N蛋白具有良好的抗原性,因而有望利用该重组蛋白开发为试剂盒应用于临床PRRSV抗体的检测. 相似文献
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副猪嗜血杆菌重组P2蛋白的高效表达及间接ELISA方法的建立 总被引：1，自引：0，他引：1
根据GenBank中副猪嗜血杆菌(HPS)的P2蛋白基因序列,设计合成1对特异性引物,用P2-PCR方法从HB070214株中克隆了1 077bp的P2蛋白基因。将P2蛋白基因亚克隆到原核表达载体pET-32a中构建了重组表达质粒pET-P2,在IPTG的诱导下成功表达了相对分子质量约为60 500的可溶性融合蛋白P2-His;经SDS-PAGE和凝胶薄层扫描分析,融合蛋白的表达量约占细菌总蛋白量的30%。将融合蛋白加入镍琼脂糖凝胶树脂层析柱,经300mmol/L咪唑磷酸盐缓冲液洗脱,其纯度可达92.5%。用Western blotting分析纯化后的融合蛋白,显示良好的生物学活性。利用纯化融合蛋白作包被抗原及优化ELISA反应条件,建立了可检测抗HPS P2蛋白抗体的间接ELISA方法(P2-ELISA),并用所建立的P2-ELISA与国外进口的HPS检测试剂盒Synbiocits-ELISA对196份临床送检血清进行平行检测,阳性符合率为93.4%。结果表明,本试验建立的P2-ELISA与Synbiocits-ELISA具有同样高的特异性。 相似文献
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为表达猪繁殖与呼吸综合征病毒(PRRSV)M蛋白主要抗原表位基因序列,参照GenBank中发表的PRRSV SCQ株M蛋白基因设计并合成一对特异性引物,通过PCR方法从重组质粒pMD18-T-M扩增得到缺失N端跨膜区的M蛋白基因片段dM(deleting M),将其与pMD19-T simple vector连接,经测序正确后克隆至高效原核表达载体pGEX-4T-1,得到重组表达载体pGEX-4T-1-dM,并将其转化大肠杆菌Rosetta(DE3),经IPTG于37℃诱导,PRRSV M基因获得表达。经SDS-PAGE分析,所表达的融合蛋白分子量约为35 kDa。以纯化的重组蛋白作为抗原,经Western Blot分析结果表明该重组蛋白可被PRRSV阳性血清所识别,可用于PRRSV的检测。 相似文献
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猪繁殖与呼吸综合征病毒CH-1a株核衣壳蛋白单克隆抗体的制备 总被引：5，自引：2，他引：5
本研究是利用我国PRRS病毒分离株CH-1a株浓缩的病毒抗原免疫BALB/c小鼠,经3次免疫后,取脾细胞与SP2/0骨髓瘤细胞进行融合,同时以PRRS病毒作为抗原进行间接ELISA检测,共筛选到22个阳性细胞株.分别经过3轮单克隆后,最终得到了16株能稳定分泌抗体的单细胞克隆株.利用IDEXX-ELISA检测试剂盒和IFA试验对16株单克隆抗体进行鉴定,结果证实所获得的16株细胞分泌的抗体都是针对PRRSV的特异性抗体.将此16株单克隆抗体分别与用原核系统表达的PRRSV N蛋白、rtM和rtGP5蛋白进行特异性ELISA检测试验,结果表明16株单克隆抗体都仅识别表达的N蛋白,而与其它两种蛋白不发生反应,说明所获得的16株单克隆抗体均为抗PRRSV核衣壳蛋白的.单克隆抗体亚型鉴定试剂盒的鉴定结果显示,除01MAb为IgG2b、N1H11为IgG2a外,其余的单克隆抗体均为IgG1,而且所有单克隆抗体的轻链均为к链.至此证实我们获得了16株PRRV N蛋白单克隆抗体,这将为今后建立更为灵敏的检测PRRS病原的特异性诊断方法、分析PRRSV核衣壳蛋白的功能及鉴定B细胞抗原表位奠定重要基础. 相似文献
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猪链球菌2型人源株MRP的表达及其单克隆抗体的制备与应用 总被引：1，自引：0，他引：1
将猪链球菌2型(SS2)人源株Habb mrp基因进行截短修饰后,克隆于pGEX4T-2载体中,转化大肠杆菌诱导表达约61 000的融合蛋白MRP-GST,该蛋白经凝血酶作用去除重组蛋白中的GST标签,得到约35 000纯化的MRP.Western blotting证实MRP可被SS2阳性血清特异性识别.以纯化的MRP抗原免疫BALB/c小鼠,取免疫鼠脾细胞与骨髓瘤细胞SP2/0融合,间接ELISA进行筛选获得了6株能稳定分泌抗MRP特异性单抗的细胞株.特异性试验表明该6株单抗与SS2的另外2种蛋白、大肠杆菌均不发生交叉反应.以2138单抗腹水和SS2多克隆抗血清建立夹心ELISA对71株标准菌株和122株猪链球菌野毒株进行MRP的表征鉴定,标准株检测结果与背景符合率为为97.2%(69/71),其中34株标准血清型菌株检测的符合率为100%.表明该方法可用于猪链球菌的快速诊断和流行病学调查. 相似文献
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猪繁殖与呼吸综合征病毒核衣壳蛋白的原核表达及其单克隆抗体的制备 总被引：1，自引：1，他引：0
试验通过RT-PCR法扩增出猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV)核衣壳蛋白(N蛋白)基因并克隆到原核表达载体pet-30a中,转入到BL21感受态细胞,经IPTG诱导表达,使用SDS-PAGE和Western blotting分析表达产物的特性。利用Ni柱纯化该重组蛋白,采用NC膜皮下包埋法免疫Balb/C小鼠,取免疫后的脾细胞与SP2/0细胞进行融合后筛选杂交瘤细胞株,检测杂交瘤细胞株的特性并制备单克隆抗体。SDS-PAGE和Western blotting结果表明,该重组蛋白表达正确,约为20 ku,能被抗PRRSV的阳性血清特异性识别。超声后用Ni柱纯化,经SDS-PAGE分析可得单一的目的条带。NC膜皮下包埋法免疫效果良好,通过细胞融合、ELISA筛选获得3株能稳定分泌抗PRRSV N蛋白抗体的杂交瘤细胞株(H7、F7、C8),制备出高特异性的针对N蛋白的H7单抗。IFA与Western blotting结果显示制备的单抗可与病毒N蛋白产生特异性反应。亚型鉴定为IgG2b型,染色体分析证实杂交瘤细胞染色体数目正确。结果成功实现了N蛋白的原核表达,并获得高特异性的单克隆抗体,为PRRSV N蛋白抗原的检测奠定基础。 相似文献
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猪生殖与呼吸综合征病毒CH-1a株GP5蛋白基因的表达及其单克隆抗体的制备 总被引：5，自引：1，他引：5
将猪生殖与呼吸综合征病毒（PRRSV）CH-1a株GP5蛋白基因信号肽部分删除后克隆于pGEX-6p-1．载体上，并在大肠埃希氏菌中进行表达。经SDS-PAGE电泳分析发现，融合表达的rtGP5蛋白约42ku，将融合蛋白rtGP5用PRRSV阳性血清进行Western-blotting分析，证实所表达的融合蛋白能被其特异性识别。收获融合表达产物rtGP5，按50pg／只的剂量与等量弗氏佐剂乳化，经腹腔免疫BALB／c小鼠3次后，取脾细胞与SP2／0骨髓瘤细胞进行融合，分别以融合蛋白rtGP5和GST蛋白为抗原，通过间接ELISA方法对融合细胞的上清液进行检测，筛选阳性克隆，结果得到了15株能稳定分泌抗rtGP5蛋白抗体的阳性细胞克隆株。经间接免疫荧光检测发现，获得的15株单克隆抗体都能与PRRSV感染细胞产生特异性荧光。15株单克隆抗体亚型鉴定结果显示均为IgG1型，且轻链均为k链。 相似文献
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基于重组猪囊虫18 ku抗原的间接ELISA方法的建立 总被引：1，自引：0，他引：1
利用反转录聚合酶链式反应(RT-PCR)从猪囊尾蚴中克隆到18 ku蛋白基因,将扩增产物与pGEM-T Easy载体连接后测序分析.将目的基因亚克隆至表达载体,构建重组质粒pGEX-CE18,经转化大肠杆菌BL21 (DE3)后诱导表达,用SDS-PAGE和Western-blot分析表达产物.表达的目的蛋白纯化后作抗原建立检测猪囊虫抗体的重组蛋白间接ELISA方法.结果表明,18 ku蛋白基因在大肠杆菌中成功表达,表达产物约为35 ku的融合蛋白,并能被猪囊虫感染血清识别.经薄层扫描分析,表达量占菌体蛋白总量的28%.与商品化ELISA试剂盒平行检测178份阳性血清样品,二者的符合率为98.83%,说明建立的重组蛋白ELISA方法可用于猪囊虫病的诊断. 相似文献
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The relationship between dry matter (DM) degradation and crude protein (CP) degradation in the dairy cow's rumen was determined with a view to defining the protein value of feeds for ruminants. The nylon bag technique was applied for these studies. For all the feeds investigate (green fodder and preserves from cocks-foot, ryegrass, alfalfa/grass and meadow grass, as well as alfalfa, extracted soybean meal) a significantly positive relationship was found to exist between the levels of DM and CP degradation (r = 0.73 to 1.0). The regression coefficient b1 (CP degradation as regressor) was found to average 0.87. The positive relationship between DM degradation and CP degradation implies that microbial protein amount and unfermented feed protein at the duodenum are negatively correlated. Model calculations show that, on account of the compensation between microbial protein and feed protein at the duodenum, in feeds with a CP concentration below 200 g/kg DM, the extent of ruminal protein degradation does not exert a marked influence on duodenal protein passage. The partial calculation of the duodenal protein supply on the basis of undegraded feed protein and microbial protein, as practiced in the new models of protein evaluation, leads to systematic errors unless the relationship between DM degradation and CP degradation is considered. 相似文献
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Spielmann J Kluge H Stangl GI Eder K 《Journal of animal physiology and animal nutrition》2009,93(4):400-409
This study was performed to assess the effects of potato protein and fish protein on concentrations of lipids in plasma and lipoproteins and the expression of genes involved in lipid metabolism in pigs used as an animal model. Therefore, 27 young male pigs with an average body weight of 22 kg were fed diets supplemented with protein extracted from potatoes (containing 849 g protein/kg dry matter), Alaska Pollack fillet as a source of fish protein (containing 926 g crude protein/kg dry matter) or casein which was used as control, for 3 weeks. Diets were formulated to supply identical amounts of each protein to the pigs by the three protein sources, namely 116 g/day in first week and 150 g/day in the second and third week. Pigs fed potato protein had lower concentrations of cholesterol in plasma and LDL than pigs fed casein (p < 0.05); no effect was observed on concentrations of HDL cholesterol and triglycerides. Pigs fed fish protein had lower cholesterol concentrations in plasma, LDL and HDL, and lower triglyceride concentrations in triglyceride-rich lipoproteins than pigs fed casein (p < 0.05). mRNA concentrations of genes involved in bile acid synthesis and cholesterol uptake were higher in pigs fed fish protein than in pigs fed casein (p < 0.05); no effect on these genes was observed in pigs fed potato protein. Expression of genes involved in lipogenesis and fatty acid oxidation was not altered by fish protein. In conclusion, this study shows that fish protein and potato protein lower plasma cholesterol concentrations in pigs. The hypocholesterolaemic effect of fish protein might be in part caused by a stimulation of bile acid synthesis; the reason for the hypocholesterolaemic effect of potato protein requires further elucidation. 相似文献
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1. Experiments were conducted to investigate whether or not varying dietary protein intake affects whole-body protein turnover rates in young chicks. 2. Seven-d-old single comb White Leghorn male chicks were fed on diets with protein concentrations of 0, 100, 200 or 400 g/kg diet under conditions of ad libitum or equalised feeding. At the end of the experiments, the rate of protein synthesis and protein degradation in the whole body were measured in vivo. 3. The results showed that both fractional and absolute rates of protein synthesis increased with increasing dietary protein up to 200 g/kg; above this concentration they remained almost constant when feeding was ad libitum. 4. Similar responses were found with equalized feeding except that a significant reduction in protein synthesis was found when dietary protein was increased from 200 to 400 g/kg diet. 5. Less sensitive and almost parallel changes in protein degradation rates were found. 6. It was concluded that adaptation to varied dietary protein intake occurred primarily through changes in protein synthesis, accompanied by parallel alterations in protein degradation in the whole body. 相似文献
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A. S. Green J. J. Ramsey C. Villaverde D. Asami A. J. Fascetti 《Journal of animal physiology and animal nutrition》2008,92(2):219-219
It is well-accepted that cats require more dietary protein than omnivores and herbivores. Work on hepatic enzyme activities showed that cats lack the ability to regulate the urea cycle enzymes in response to the dietary supply of protein. It was thus hypothesized that the high protein requirement of cats is due to an inability to regulate these enzymes, limiting adaptation to a low protein diet. We used indirect respiration calorimetry to assess the in vivo ability of cats to adapt substrate oxidation to different levels of dietary protein, including one below their protein requirement. In random order, eight cats consumed each of four semi-purified diets containing 7.7% (LP), 14.6% (AP), 27.3% (MP) and 51.1% (HP) of ME from protein. Cats consumed each diet for at least 14 days and then completed a 5-day nitrogen balance trial and at least 2, 12-hour indirect calorimetry measurements. The data were analyzed by anova using the Mixed procedure of SAS and are expressed as mean ± SEM. There was a significant effect of diet on protein oxidation (p < 0.0001), measuring 9.8 ± 0.5%, 13.4 ± 0.9%, 23.5 ± 0.8% and 49.0 ± 1.8% of total energy expenditure on the LP, AP, MP and HP diets, respectively. The ratio of protein oxidation/protein intake was significantly higher with the LP diet (1.27 ± 0.07) than the other three diets (AP, 0.92 ± 0.06; MP, 0.86 ± 0.03; HP, 0.96 ± 0.04; p < 0.0001), indicating a net loss of protein on the LP diet. Thus, cats adapted to a wide range of dietary protein concentrations, but were unable to fully adapt to the LP diet. 相似文献
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乳猪能否顺利断奶是养猪业成功的关键.乳猪消化系统的发育尚未完善,需要易消化、质量高的蛋白原料.因此,高质量、易消化的乳蛋白和血浆蛋白等常被用作乳猪开食料的蛋白原料. 相似文献