家蚕蛹到蛾期蛋白SDS—PAGE电泳观察

蛹到蛾期蛋白SDS-PAGE电泳约有17条左右的蛋白带,蛋白组分有一定变化。主要观察到:1)分子量约为220kD的蛋白带在化蛹第2天出现,并随着蛹期发育至羽化,其浓度不断的增加。2)分子量约为70kD的蛋白带,化蛹第1天是一条带,第2天至羽化其蛋白组分有变化。3)分子量约为29kD的蛋白带随着蛹期发育到羽化,浓度逐渐减弱。     

用SDS—PAGE分析鸡毒霉形体广西分离株的结构蛋白

应用SDS－PAGE对鸡毒霉形体（Mycoplasma gallisepticum,MG)4个标准株和5个广西分离株的结构蛋白进行了比较分析。结果，在凝胶电泳图谱中10－100ku蛋白分子质量之间，F株缺少87ku蛋白带，Y3株在最靠近87ku蛋白带的上方和下方各缺少1条蛋白带，H2株和Y2株缺少64ku蛋白带，CH株缺少29ku蛋白带，97、75、43ku处的蛋白带为4个MG标准株和5个MG分离株所共有。表明9个MG供试菌株的结构蛋白都存在一定的差异，广西MG分离株的结构蛋白呈现多样性。     

8株鳖源变形杆菌外膜蛋白的比较

从 1 2批送检病鳖体内分离出 8株细菌 ,经形态学检查、生理生化特性测定、致病性测定和血清学鉴定 ,确定 7株为普通变形杆菌 ,1株为奇异变形杆菌。进一步采用十二烷基硫酸钠 (SDS)破菌法提取 8株鳖源变形杆菌的外膜蛋白 (OMP)进行SDS PAGE电泳 ,比较分析细菌OMP型。结果显示奇异变形杆菌的OMP型由 3条相对分子质量范围为 3 0× 1 0 4～4 3× 1 0 4的主要蛋白带组成 ;7株普通变形杆菌的主要外膜蛋白相对分子质量范围为 3 0× 1 0 4～6 7× 1 0 4,分属 2个OMP型 ,其中 4个分离株属OMP1型 ,由 4条主要蛋白带组成 ;其余 3个分离株属OMP2型 ,由 7条主要蛋白带组成。表明不同种变形杆菌的OMP型差异较大 ,同种不同株变形杆菌的OMP型相似 ,但蛋白带的迁移率及颜色深浅在菌株间仍有差异。此外 ,发现相对分子质量约为 4 3× 1 0 4的一条外膜蛋白带为所有菌株所共有 ,可能是变形杆菌属特异性抗原     

用单克隆抗体对鼻疽杆菌和类鼻疽杆菌表面抗原的免疫印迹分析

对鼻疽杆菌M27株与类鼻疽杆菌H4、H103、H146、H152株超声波粉碎后的可溶性抗原作了SDS—PAGE和免疫印迹分析.结果表明,鼻疽杆菌和类鼻疽杆菌特异性MCAb 2D_4与所有实验菌株都有反应,在M27、H4、H103、H146、H152中都有1条分子量为107000的抗原蛋白带,为鼻疽杆菌和类鼻疽杆菌共同抗原带;而类鼻疽杆菌特异性McAb 3A_1只与类鼻疽杆菌H4、H103、H146、H152,分子量为28000的类鼻疽杆菌特异抗原带起反应,不与鼻疽杆菌的抗原带反应.尽管SDS—PAGE中显示M27与H4有多条分子量相同条带,但缺少3A_1对应的特异蛋白带.     

绵羊肉孢子虫可溶性蛋白抗原的特异性初探

应用十二烷基硫酸钠—聚丙烯酰胺凝胶电泳(SDS—PAGE)分析绵羊肉孢子虫囊壁和不同种肉孢子虫滋养体蛋白带,结果表明:肌肉中消化分离出的羊犬肉孢子虫滋养体和从食道壁上挑出的巨肉孢子虫滋养体均出现三条电泳蛋白带;巨肉孢子虫全虫体可溶性蛋白出现五条蛋白带;巨肉孢子虫囊壁可溶性蛋白出现两条电泳蛋白带;微小住肉孢子虫滋养体可溶性蛋白出现一条电泳蛋白带。用不同种的滋养体可溶性蛋白抗原和巨肉孢子虫囊壁抗原做IHA试验表明:用羊犬肉孢子虫和巨肉孢子虫滋养体可溶性蛋     

1株鸭源新城疫病毒HN蛋白潜在抗原表位的分析

为了比较鸭源新城疫病毒(NDV)XZ株的HN蛋白的抗原表位与La Sota疫苗株间的差异,试验采用可塑性、亲水性、表面可能性、抗原性、二级结构等5项参数指标来综合预测鸭源NDV XZ株与疫苗株La Sota株HN蛋白的潜在B细胞抗原表位。结果表明:鸭源NDV XZ株HN蛋白预测抗原表位有26个,La Sota株HN蛋白预测抗原表位有25个。与La Sota株HN蛋白相比,多出2个抗原表位,缺失1个抗原表位。这3个表位的变化可能会对病毒识别细胞膜上的唾液酸受体及机体的免疫应答造成影响。两者HN蛋白都只有1个跨膜区域,位置接近。说明鸭源NDV XZ株HN蛋白与La Sota株HN蛋白相比,可能具有相近的抗原性,还需要进一步验证。     

NDV江苏分离株F蛋白潜在抗原表位分析

从可塑性、亲水性、表面可能性、抗原性、二级结构等5项参数指标来综合预测1株基因Ⅶ型新城疫病毒(Newcastle disease virus,NDV),NDV JSXZ株F蛋白的潜在B细胞抗原表位,并比较其与疫苗株La Sota抗原表位的差异,同时对F基因序列进行分析。综合预测显示:NDV JSXZ株F蛋白预测抗原表位有23处;La Sota F蛋白预测抗原表位分别有19处。与La Sota F蛋白相比,NDV JSXZ毒株在222~228、260~267、308~314、401~413位氨基酸处多出4处抗原表位,而在29~37、104~113、482~488位氨基酸有3处抗原表位不同。其中29~37、104~113aa这2处抗原表位位于裂解位点之前,可能对病毒蛋白水解酶的敏感性、毒力等造成影响。应用Protein-TM-Plot软件预测到NDV JSXZ株F蛋白有3个跨膜区域,分别位于9~27,115~143,497~525位氨基酸处,与La Sota株的三个跨膜区位置接近。结果提示,NDV JSXZ株F蛋白与La Sota株F蛋白相比,可能具有相近或更强的抗原性。     

四株新城疫病毒流行株F基因的遗传变异分析

设计并合成了两对寡核苷酸引物，P1和P2用于扩增新城疫病毒（NDV）全长F基因，P3和P4用于扩增NDV部分F基因。通过RT-PCR一次性扩增了NDV四平株和长春株的全长F基因和青岛株、昌黎株的部分F基因。将这些基因分别克隆后，进行了序列测定和同源率、致病性、疏水性、抗原性和系统发育等比较分析。结果表明，四平株和长春株F基因同为1762bp,编码553个氨基酸。两种F基因推导的氨基酸序列的疏水性相似，都具有3个强疏水区，但亲水性有差异，抗原表位也有明显的不同，说明四平株和长春株F蛋白的抗原性不同。4株NDV F基因序列（1-813bp)和推导的氨基酸序列（261aa)与我国台湾和国外有代表性的NDV F基因相同区域进行了比较和系统发育分析。总体上，核苷酸序列同源率在82.9%-97.5%之间，推导的氨基酸序列同源率在85.8%-97.7%之间，长春株与La Sota、Texas GB、Beaudette在同一亚群，四平株、昌黎株在同一亚群，青岛株为另一亚群。四平株、青岛株和昌黎株F蛋白裂解位点区（112-117aa)的氨基酸序列与强毒株在这一区域的序列相符，证明这3个NDV毒株是强毒株，长春株F蛋白裂解位点区的氨基酸序列与弱毒株的序列相符，证明是弱毒株。     

疫苗免疫压力下新城疫病毒的动态演化

国内大量新城疫病毒(NDV)血凝素基因F和HN的测序表明,NDV主要免疫原HN和F基因与生产中广泛应用的经典疫苗La Sota株的核苷酸同源性不足80%,而NDV流行株之间则高达94.4%～100%,从分子遗传学角度证实了VIId型NDV是导致新城疫免疫失败的重要原因。HI交叉抑制和鸡胚中和试验等则从抗原性的角度证实了NDV在免疫压力下抗原性的变化,尽管NDV不同毒株间抗原性变异的程度已出现明显差异,但仍局限在同一个血清型中。     

温度对嗜水气单胞菌外膜蛋白表达的影响

提取嗜水气单胞菌J-1、Y-1、F-155 株的外膜蛋白(OMP)作SDS-PAGE,结果显示,它们的电泳图谱属于同一模式,均含有22 000、31 000、38 000、43 000 和50 000 等5 条主要OMP带。温度影响嗜水气单胞菌OMP的表达,28℃培养时表达的OMP以43 000 者为最多;而37℃培养时表达最多的是38 000 的条带。J-1株37℃培养时对鲫鱼和小鼠的LD50分别为3.4×106、2.5×106 CFU;28℃时则为8.7×105、1.8×105 CFU,表明28℃培养时J-1 株毒力较强。随着OMP在载样缓冲液中煮沸时间的增加,主要蛋白带的浓度降低,相对分子质量小的蛋白带增多。免疫转印显示,这些蛋白带均能与J-1 株OMP多抗呈显色反应。抗原性分析表明,J-1 株OMP与HEC毒素之间无抗原性交叉。     

Structural proteins of classic and variant strains of infectious bursal disease viruses.

Structural polypeptides of six tissue-culture-origin (BGM-70 continuous cell line) infectious bursal disease viruses representing classic and variant strains of serotype 1 and one serotype 2 strain were analyzed and compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Additionally, two of the variant strains were propagated in vivo in bursa of Fabricius and compared with those grown in cell culture. Differences among the structural proteins of serotype 1 viruses were minor and probably of no value in differentiating these viruses. However, distinct differences were observed between serotype 1 and 2 viruses. The bursa-derived viruses were different from those propagated in cell culture in molecular weights and in proportions of the proteins. The bursa-derived strains had protein migration patterns similar to those described for tissue-culture-incomplete virus particles.     

Preparation and characterisation of envelope proteins from Haemophilus pleuropneumoniae

Envelope proteins of Haemophilus pleuropneumoniae were extracted by 3 methods and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Three major envelope proteins (45,000 Mr, 41,000 Mr, 31,500 Mr) were distinguished in sonicated cell envelopes together with minor proteins. Using selective solubilisation with sodium lauryl sarcosinate or Triton X-100, outer membrane proteins were distinguished from those of the cytoplasmic membrane. Extraction into LiCl produced a similar profile, but the 41,000 Mr and 31,500 Mr bands were present in reduced amounts. Extraction into saline at 60 degrees C produced a grossly different pattern, with a major band at 20,000 Mr. All 3 major envelope proteins were shown to be heat-modifiable, and the 31,500 Mr band was found to be the non-heat-modified form of a 43,000 Mr protein, which showed similar properties to the Protein d of H. influenzae which is related to the OmpA protein of E. coli K-12. The 45,000 Mr major protein was also weakly associated with the peptidoglycan in SDS/Triton at low temperature.     

Polyacrylamide gel electrophoresis of whole-cell preparations of Rhodococcus equi.

The whole-cell proteins of ten strains of Rhodococcus equi isolated from horses, pigs, or humans, including the type strain ATCC 6939, were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The protein profiles of seven different capsular serotypes and the type strain were very similar when bacteria were cultured under the same conditions. Protein profiles were largely unaffected by incubation at two temperatures (30 degrees C, 37 degrees C) or times (12 h, 48 h). There were generally minor differences in protein profiles between strains grown in different media (brain heart infusion, nutrient, minca broths, tryptic soy-blood agar) with the marked exception of a prominent diffuse 17.5 kd protein which was expressed in nutrient broth. This protein was not produced by the type strain and was lost on repeated passage in vitro (50th, 100th passage) in two of three other strains examined.     

猪塞尼卡谷病毒单克隆抗体的制备及鉴定

[目的] 纯化猪塞尼卡谷病毒（Seneca Valley virus，SVV）SVV-CH-HB2016毒株，并制备其结构蛋白VP1、VP2和VP3的单克隆抗体。[方法] 以蔗糖密度梯度离心法纯化的SVV-CH-HB2016病毒颗粒作为抗原，免疫BALB/c小鼠，取脾细胞与骨髓瘤细胞（SP2/0）进行细胞融合。通过间接免疫荧光试验（IFA）结合间接ELISA筛选阳性细胞株，制备能特异性分泌针对结构蛋白的杂交瘤细胞株。采用Western blotting和IFA方法分别检测单克隆抗体与重组表达蛋白及天然结构蛋白的反应性，并对单克隆抗体的病毒中和保护效果进行测定。利用空斑试验和实时荧光定量PCR方法探究中和性单克隆抗体对SVV-CH-HB2016毒株吸附过程的影响，最后用抗体相加试验来分析14株单克隆抗体的抗原表位。[结果] 在蔗糖密度梯度为5%~45%（W/V）时获得了纯度较好、浓度较高的SVV-CH-HB2016毒株结构蛋白，免疫小鼠血清抗体效价均达到了1：12 800，成功制备了17株能稳定分泌特异性单克隆抗体的杂交瘤细胞株。经验证14株单克隆抗体能与重组结构蛋白发生Western blotting反应，17株单克隆抗体能与病毒发生IFA作用；2G6、4A3和4C11 3株单克隆抗体对SVV-CH-HB2016毒株感染的BHK-21细胞具有明显中和保护作用，也能有效抑制SVV-CH-HB2016毒株对293T细胞的吸附。经分析发现，除1F5与2E1、4B8与4F11外，其余10株单克隆抗体分别针对不同抗原表位。[结论] 本研究初步建立了SVV的纯化方法，制备了17株特异性针对SVV-CH-HB2016毒株的单克隆抗体，为后期进一步开展SVV全病毒灭活疫苗的研发、ELISA检测方法的建立及保护性抗原表位的鉴定奠定了基础。     

鸡毒支原体株间结构蛋白及其抗原性变异的比较研究

本研究以ＳＤＳ－ＰＡＧＥ及Ｗｅｓｔｅｒｎ Ｂｌｏｔ技术,应用鸡毒支原体国外强毒株Ｓ６、标准株ＰＧ３１,疫苗株Ｆ,北京分离 ＢＧ４４Ｔ、ＮＢ７２特异性多克隆抗血清对以上五株及疫苗株Ｖ、北京分离株Ｃ的结构及其抗原性进行了比较结果表明ＭＧ结构蛋白及其抗原性存在着株间的多样性和一定相似性,其中以Ｆ与Ｓ６、ＢＧ４４Ｔ与ＰＧ３１、ＮＢ７２与Ｃ更为接近,可能具有同源性。ＳＤＳ－ＰＡＧＥ显示出了ＭＧ株间结构蛋白微     

Protein electrophoretic pattern of Pasteurella haemolytica.

Electrophoresis in polyacrylamide gels of the constituent proteins of the 12 serotypes and an untypable strain of Pasteurella haemolytica showed a pattern of bands that divided the group into two. This division conformed to the A and T biotype groupings of Smith (1959) although the serotype A9 showed only minor band difference from the three T serotypes 3, 4 and 10. It was not possible by this method to separate all the type strains from each other by the specific recognition of the patterns of protein mobilities produced.     

Identification of the antigenic components of the virulent Mycoplasma gallisepticum (R) in chickens: their role in differentiation from the vaccine strain (F)

The antibody response to different proteins of Mycoplasma gallisepticum (MG) was studied in chickens experimentally infected with virulent MG R strain. The chickens were challenged at 8 weeks of age by the intranasal route. Each cockerel received 1.3 X 10(6) colony-forming units (CFU). MG strains (R and F) were banded by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The banding pattern was distinctively different between the two strains in the range of 92.5 to 200 kilodaltons (kD). Chicken sera collected at different times following challenge were analyzed by Western blot to determine the patterns of antibodies raised to specific MG proteins (R versus F strains). Early in infection (2 weeks postchallenge), antibodies to 60-kD and 75-kD polypeptides of MG R strain were produced. Subsequently (greater than or equal to 4 weeks postchallenge), antibodies recognized a larger number of MG antigens in both strains. The immunoblot patterns remained the same in the period 8-11 weeks postinfection in each of the two strains; however, the patterns were different when the two strains were compared. The early response recognized the 75-kD protein in the R strain while it recognized the 80-kD protein in the F strain. The late response recognized the 130-kD protein and the protein slightly heavier than 200 kD in the R strain. These two bands did not appear in the immunoblot performed against the F strain of MG. Electroeluted protein of MG R strain, namely adhesin (75 kD), showed a hemagglutination activity (HA) on chicken red blood cells. With the appearance of antibodies specific to the 60-kD and 75-kD polypeptides, there was a significant rise in hemagglutination-inhibition geometric mean titer of chicken sera.     

Staphylococcus aureus seroproteomes discriminate ruminant isolates causing mild or severe mastitis

ABSTRACT: Staphylococcus aureus is a major cause of mastitis in ruminants. In ewe mastitis, symptoms range from subclinical to gangrenous mastitis. S. aureus factors or host-factors contributing to the different outcomes are not completely elucidated. In this study, experimental mastitis was induced on primiparous ewes using two S. aureus strains, isolated from gangrenous (strain O11) or subclinical (strain O46) mastitis. Strains induced drastically distinct clinical symptoms when tested in ewe and mice experimental mastitis. Notably, they reproduced mild (O46) or severe (O11) mastitis in ewes. Ewe sera were used to identify staphylococcal immunoreactive proteins commonly or differentially produced during infections of variable severity and to define core and accessory seroproteomes. Such SERological Proteome Analysis (SERPA) allowed the identification of 89 immunoreactive proteins, of which only 52 (58.4%) were previously identified as immunogenic proteins in other staphylococcal infections. Among the 89 proteins identified, 74 appear to constitute the core seroproteome. Among the 15 remaining proteins defining the accessory seroproteome, 12 were specific for strain O11, 3 were specific for O46. Distribution of one protein specific for each mastitis severity was investigated in ten other strains isolated from subclinical or clinical mastitis. We report here for the first time the identification of staphylococcal immunogenic proteins common or specific to S. aureus strains responsible for mild or severe mastitis. These findings open avenues in S. aureus mastitis studies as some of these proteins, expressed in vivo, are likely to account for the success of S. aureus as a pathogen of the ruminant mammary gland.     

Characterization of the structural proteins of porcine epizootic diarrhea virus, strain CV777

Pig epizootic diarrhea virus cannot be grown in cell culture; for its characterization, intestinal perfusate material from a pig infected with the strain CV777 had to be used. In isopyknic sucrose gradients, a peak of virus-specific ELISA activity was detected at a density of 1.17 g/ml. Using immunoprecipitation of radioiodinated-purified virus material followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 3 proteins of low molecular weight (20,000 to 32,000 daltons [D]) were found; after blotting nitrocellulose and glycoprotein identification with concanavalin A and horseradish peroxidase, 1 of the proteins (23,000 D) gave a signal. Another protein of 58,000 D was encountered, which was the only protein binding an RNA probe. Finally, a protein of 85,000 D was visible, associated with minor bands of about 110,000 and 135,000 D in most experiments. Using the concanavalin A-blotting technique, the same bands were visualized. The demonstration of a polydisperse cluster of proteins from 20,000 to 32,000 D (of which at least 1 is glycosylated), of glycosylated proteins from 85,000 to 135,000 D, and of an RNA-binding protein of 58,000 D is taken as structural evidence that pig epizootic diarrhea virus should be classified with the Coronaviridae, irrespective of the apparent lack of an antigenic relationship with other members of that family.     

Immunoblotting study of the antigenic relationships among eight serogroups of Leptospira.

Seven strains of Leptospira interrogans belonging to seven different serogroups, and one strain of Leptospira biflexa were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with gradient gels and immunoblotting with hyperimmune rabbit sera raised against each strain. The molecular masses of the proteins were calculated with a polynomial regression model. The SDS-PAGE patterns of the L. interrogans strains were similar and characterized by 24 common bands. This profile was not found for L. biflexa. The immunoblots obtained either with the seven anti-L. interrogans sera or the anti-L. biflexa serum allowed a clear distinction between the two species. Taken as a whole, the L. interrogans strain patterns revealed by the seven anti-L. interrogans sera were similar, sharing eight common major bands. A serovar- or serogroup-specific antigenic zone, ranging from 21 to 26 kDa, was also identified.