共查询到18条相似文献，搜索用时 218 毫秒
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为建立从患有非典型新城疫的蛋鸡组织细胞中提取、纯化热休克蛋白HSP70抗原肽复合物的方法,采取三步蛋白纯化法从非典型新城疫蛋鸡肝脏组织中提取、纯化HSP70抗原肽复合物.经分级硫酸铵盐析后,依次用ConA-Sepharose亲和层析和DEAE-Sephacel离子交换层析分离纯化.所得蛋白经SDS-聚丙烯酰胺凝胶电泳和Western blotting进行蛋白分子量及性质鉴定,Bradford法测定蛋白浓度.分离、纯化得到的蛋白经SDS-PAGE、银染鉴定为单一带,分子量为70ku;Western blotting结果证实为HSP70.每克新鲜肝脏细胞最终获得90～110 μg HSP70.使用本分离纯化方法可获得高纯度HSP70抗原肽复合物. 相似文献
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利用超滤法从羊和猪胎盘中提取出了胎盘肽,采用改良的加脲Tricine-SDS-PAGE电泳法测定羊胎盘肽的分子量为4,386Da;利用紫外光扫描得其最大吸收峰分别为225.4 nm和209.6 nm;并通过建立体外抑制兔淋巴细胞模型,利用MTT比色法测定所提取胎盘肽的免疫调节作用,结果显示:羊和猪胎盘肽均可使顺铂抑制的兔外周血T淋巴细胞的转化率显著提高,其中羊胎盘肽以1:102、1:103和1:104三组效果最为明显(P<0.01);而猪胎盘肽以1:102、1:103两组效果最为明显(P<0.01)。 相似文献
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《畜牧与兽医》2020,(10)
为建立新的禽白血病诊断方法,以临床分离得到的JL-2株禽白血病前病毒cDNA为模板,用PCR技术扩增gp37基因,测序后进行序列分析,并构建原核表达质粒pET-gp37,在大肠杆菌BL21(DE3)中经IPTG诱导表达,SDS-PAGE及Western blot鉴定并纯化。结果显示:gp37基因全长591 bp,编码197个氨基酸,分子量22 kDa,无信号肽,为疏水性蛋白;PCR及双酶切鉴定表明,重组原核表达质粒pET-gp37构建成功;经SDS-PAGE及Western blot鉴定,表达蛋白分子质量与理论值一致,均为22 kDa,并以可溶形式表达,纯化后纯度达98%以上。结论:成功表达和纯化了gp37蛋白,为预防禽白血病病毒的感染及建立新的禽白血病诊断方法提供了参考。 相似文献
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本文简要论述了微生物发酵法和酶解法生产饲用小肽产品的方法,从蛋白原料、生产工艺及操作参数、后加工处理方法等各个方面论述了小肽产品品质的影响因素,并从常规指标、小肽含量、小肽分子量分布、抗营养因子几个方面,扼要论述了饲用小肽产品检测方法和质量甄别办法,便于饲料企业和广大用户如何检测和甄选饲用小肽产品。 相似文献
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抗菌肽（ABP）又称为抗微生物肽（AMP）、宿主防御肽（HDPs）、肽抗生素，是动物免疫防御系统在外界条件刺激下，产生的一类对抗外原陛病原体致病作用的具有免疫活陛的多肽，是动物非特异性免疫防御系统的一个重要组成部分。依据其物种来源的不同，被分别命名为防御素、蛙皮素和蜂毒素等。抗菌肽有着分子量小、活性强、功能广泛、杀菌谱广等特点。抗菌肽的分子量一般在4kDa左右，肽链由12～50个氨基酸残基组成， 相似文献
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[目的]为了深入研究杂合抗菌肽Melitten-cathelicidin_BF的理化性质,为抗菌肽开发利用提供新的思路。[方法]本研究将蜂毒肽（Melitten）和金环蛇抗菌肽（Cathelicidin_BF）进行融合构成新的多肽,利用在线生物分析软件进行了全面系统的生物信息学分析。[结果]杂合肽Melitten-cathelicidin_BF一级结构,含有56个氨基酸残基,包括14种氨基酸,分子量为6.47 kDa,理论等电点为12.20,为亲水性蛋白,有5个糖基化和2个磷酸化修饰位点。二级结构主要元件为α螺旋和无规则卷曲。[结论]该杂合肽无信号肽、无跨膜区,且该杂合肽为阳离子肽,该杂合肽为蛋白酶抑制剂类肽。 相似文献
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Immunological characterization of breakdown peptides of the 104 kilodalton hemolysin of Actinobacillus pleuropneumoniae serotype 1 总被引：1，自引：0，他引：1
The 104 kilodalton (kDa) hemolysin of Actinobacillus pleuropneumoniae serotype 1, strain CM-5 was precipitated from RPMI-1640 culture supernatant using ammonium sulfate to 80% saturation. In immunoblots, a rabbit polyclonal antiserum against the 104 kDa hemolysin protein, recognized not only the original 104 kDa monomeric form of the hemolysin but other proteins in the crude antigen mixture ranging in molecular mass from 43 to greater than 125 kDa. The antiserum was able to crosslink these proteins to active hemolysin in RPMI-1640 culture supernatant resulting in bands of hemolysis in blood agar used in a contact assay. Corresponding to these bands of hemolysis, denatured peptides with molecular masses of 51, 85, 104 and greater than 125 kDa were excised and injected into rabbits. In immunoblots, the resultant antibodies recognized the injected peptide and the monomeric 104 kDa protein. However, only the rabbit antisera produced against the 104 and 125 kDa proteins contained antibodies which neutralized the active 104 kDa hemolysin in culture supernatant. These results indicate that (i) the 104 kDa protein hemolysin can exist in a higher molecular weight aggregate (greater than 125 kDa) but can also break down to peptides which have molecular masses smaller than the 104 kDa parent molecule and (ii) while several epitopes are present in the hemolysin molecule, there seems to be a restricted number of antigenic determinants responsible for inducing neutralizing antibodies and these seem to reside only in the 104 kDa parent molecule. This may have consequences, in terms of vaccine development, for the control of pleuropneumonia in swine herds. 相似文献
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试验旨在分离纯化多黏类芽孢杆菌（Paenibacillus polymyxa）BLCC1-0402代谢产物中的抗菌肽,为抗菌肽制备及其制品检测提供参考。采用离心、不同分子质量卷式膜超滤浓缩、Superdex peptide 10/300GL凝胶过滤层析对多黏类芽孢杆菌发酵上清液进行逐级分离纯化,对不同时段的收集液做抑菌试验,以大肠杆菌O78标准菌株为指示菌,采用打孔法进行抑菌活性检测,比较评价分步层析效果,以Tricine-SDS-PAGE进行分子质量检测。结果显示,通过5和3 ku卷式膜超滤获得的3~5 ku组分蛋白质样品抗菌活性较强;对于3~5 ku组分经凝胶过滤层析分离纯化,纯化后的抗菌肽A3抑菌活性最强,经Tricine-SDS-PAGE小分子多肽电泳检测,已达到电泳纯,分子质量为4 ku;抑菌活性检测结果显示,该抗菌肽A3对大肠杆菌O78标准菌株具有较强的抑菌作用。同时,抗菌肽A3表现出较好的耐热性,90~100 ℃处理15 min,抑菌活性可保持在96%左右;具有较好的酸碱稳定性,在pH 2.0~9.0下,抑菌活性保持在90%以上;经胃蛋白酶作用后抗菌肽A3抑菌活性降低20%,胰蛋白酶作用后抗菌肽A3抑菌活性降低18%,蛋白酶K对抗菌肽A3的抑菌活性几乎无影响。本研究结果表明,分离得到的抗菌肽A3是一种对大肠杆菌O78具有抑菌活性的新型抗菌肽,具有一定的开发潜力,可为下一步抗菌肽的结构分析、理化特性分析等深入研究提供一定参考依据。 相似文献
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构建表达抗菌肽Cecropin AD蛋白的真核重组表达质粒pGAPZα-A-CAD,并转化酵母菌Pichia pastoris X-33,Zeocin抗性筛选阳性重组子,通过PCR鉴定、Tricine-SDS-PAGE分析和琼脂孔穴扩散法筛选获得抗菌肽CAD组成型表达菌株,并采用pH-溶氧监控策略进行工程菌Pichia pastoris X-33/pGAPZα-A-CAD的50 L中试发酵研究。结果表明,本实验成功实现了抗菌肽CAD在毕赤酵母Pichiapastoris X-33中的组成型表达pGAPZα-A,Tricine-SDS-PAGE分析显示在4.0 kDa处有明显的目的条带,工程菌经50 L发酵罐培养48 h后收获的上清液,100℃热处理10 min后,对金黄色葡萄球菌ATCC25923、大肠杆菌ATCC25922等均有明显的抑杀作用。抗菌肽的组成型表达方法在本研究中得到了成功应用,中试研究为以后的规模化生产和应用奠定了基础。 相似文献
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Puchalski A Dec M Wernicki A Urban-Chmiel R Gieral A 《Polish journal of veterinary sciences》2010,13(1):121-127
Iron-regulated outer membrane proteins (IROMPs) of P. multocida serotype A3, which function as receptors for complexes containing iron ions, are induced by iron deficiency in the bacterial growth environment. Analysis of an electrophoresis image of proteins isolated from bacteria grown on medium supplemented with 2,2'-dipyridyl revealed expression of 16 new proteins that were not noted in the case of the bacteria grown in standard conditions, with molecular weights from 30 to 160 kDa. Induction of IROMP expression occurred within 30 minutes after restricted iron conditions were established. In immunoblotting, distinct reactions were noted for proteins of molecular weight ranges of 25-49 kDa, 61-95 kDa, and 108-214 kDa. Proteins of the molecular weight of 68, 75 and 86 kDa were analysed using mass spectrometry and matched with the highest probability to proteins in the NCBI data base. Several dozen different proteins with similar amino acid sequences were matched to each sample. 相似文献
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J M Molina A Ruiz E Rodríguez-Ponce A C Gutiérrez J González S Hernández 《Veterinary research》1999,30(4):393-399
The adult Haemonchus contortus somatic antigens responsible for cross-reactivity have been analysed using serum samples from goat kids infected and reinfected with Teladorsagia circumcincta. Goat kids infected with T. circumcincta had similar serum ELISA values against somatic antigens of H. contortus as goats infected with H. contortus itself. Immunoblotting confirmed this extensive cross-reactivity particularly in the molecular weight range 105-29 kDa. However, peptides with high (195, 152 and 119 kDa) or low (23 kDa) molecular weight were only faintly recognized by heterologous sera. 相似文献
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In the early stages of embryonic development, many growth-promoting molecules must be provided by the maternal system. These factors may be supplied locally to the embryo, by the decidua, the placenta, or the yolk sac. In this study the growth-promoting potential of extra-embryonic coelomic fluid (EECF) and its fractions was investigated. The embryonic requirement of growth-promoting molecules may be studied by reducing the growth-supporting capacity of serum. Thus, ultrafiltration of rat serum was carried out for 8 h using Millipore filters with a molecular weight exclusion of 30 kDa. Rat embryos at 9.5 days of age were cultured for 8 days for anembryonic yolk sacs, and then EECF was collected and divided into three different molecular weight fractions by ultrafiltration. Rat embryos were cultured for 48 h in whole rat serum and the serum retenate (which has low growth-supporting capacity) in the presence and absence of EECF, its fractions, or in EECF only. Embryos grown in retenate showed severe growth retardation, and the addition of EECF significantly improved embryonic growth. The fraction which contained the molecules with molecular weight between 10 and 30 kDa had significantly more effect on embryonic development than the other fractions. This fraction of EECF was analysed by gel electrophoresis. Three of the four protein bands observed in this fraction were identified by amino-terminal sequencing as alpha-fetoprotein precursor (22 kDa), apolipoprotein A1 precursor (24 kDa) and fetal haemoglobin Y2 chain (14 kDa), none of which are likely to be responsible for the growth-promoting activity. To further investigate growth-promoting proteins, EECF was Western-blotted to nitrocellulose membranes and probed with antisera against rat prolactin, epidermal growth factor, insulin-like growth factors I and II and human placental lactogen. No immunoreactive bands were detected in the EECF, suggesting that either these proteins are not present or are present at levels too low to be detected. Although the growth-promoting effect of the EECF was demonstrated in this study, the molecules responsible remain uncharacterized. 相似文献
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Tani H Shimizu R Sasai K Baba E 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2003,65(10):1049-1056
Circulating thyroglobulin autoantibody (TgAA) was analyzed using the Western immunoblot for determination of the dominant epitopes recognized by TgAA on tryptic peptides of canine thyroglobulin (cTg) in hypothyroid dogs. TgAA was measured in hypothyroid dogs, non-hypothyroid dogs with skin diseases and clinically normal dogs. Five of the 7 hypothyroid dogs, 1 of the 8 dogs with skin diseases and 1 of the 4 normal dogs were positive for TgAA. Four of the 5 TgAA-positive hypothyroid dogs were Golden Retrievers, and 3 of them showed high antibody titers. The sera of TgAA positive-dogs reacted to several peptides, and their patterns varied from sample to sample. Sera from 3 dogs with high titers of TgAA reacted broadly to high molecular weight peptides ranging from 45 to 90 kDa. These Western immunoblot patterns of the sera were disappeared after pretreatment with sufficient amount of intact cTg. All serum samples of both TgAA positive dogs and negative controls reacted to low molecular weight peptides ranging from 15 to 20 kDa. These immunoblot patterns of the sera were not disappeared even after pretreatment with sufficient amount of intact cTg. These findings show the possibility that the epitopes recognized by TgAA depend upon individual dogs with hypothyroidism and these autoantibodies recognize conformational epitopes on the cTg molecule. 相似文献
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McCall C Hunter S Stedman K Weber E Hillier A Bozic C Rivoire B Olivry T 《Veterinary immunology and immunopathology》2001,78(3-4):231-247
Although house dust mites (HDM(s)) are important elicitors of canine allergy, the low molecular weight molecules defined as major allergens for humans do not appear to be major allergens for dogs. Western blotting of Dermatophagoides farinae (D. farinae) extracts with sera from sensitized dogs showed that the majority of animals had IgE antibodies specific for two proteins of apparent molecular weights of 98 and 109kDa (98/109kDa). The N-terminal sequences of these two proteins were identical, suggesting they were very closely related, and sequencing of internal peptides showed the protein(s) to have homology with insect chitinases. A purified preparation of 98/109kDa proteins elicited positive intradermal skin tests (IDST(s)) in a group of well-characterized atopic dogs sensitized to D. farinae, but not in normal dogs. A rabbit polyclonal antiserum raised against the purified proteins was used to immunoscreen a D. farinae cDNA library. The mature coding region of the isolated chitinase cDNA predicts a protein of 63.2kDa; sequence analysis and glycan detection blotting suggest that the molecule is extensively O-glycosylated. Monoclonal antibodies made against the purified native protein were used to localize the chitinase in sections of whole D. farinae mites. The protein displayed an intracellular distribution in the proventriculus and intestine of the mite, suggesting that it has a digestive, rather than a moulting-related, function. The high prevalence of IgE antibodies to this antigen in canine atopic dermatitis makes it a major HDM allergen for dogs, and the protein has been formally designated Der f 15. 相似文献