鹦鹉热衣原体的实验室诊断

鹦鹉热衣原体系专性细胞内寄生物能在鸡胚和易感的畜禽细胞内生长繁殖。衣原体含有两种抗原,一种是耐热的,具有属特异性;一种是不耐热的,具有种特异性。鹦鹉热衣原体除含有外膜LPS外,还含有一层主要由几种多肽组成的蛋白质外膜(M OM P)约占被膜蛋白总量的60%。M OM P在抗原的分     

沙眼衣原体鼠肺炎株外膜蛋白抗原特性的研究

用提纯的鼠肺炎株免疫Balb/C小鼠,建立了一株能分泌种特异单克隆抗体和一株能分泌型特异单克隆抗体的杂交瘤细胞株。用电泳技术和兔疫印迹试验证实,所建立的种特异单克隆抗体可与各种沙眼衣原体血清型的外膜主蛋白(MOMP)抗原相反应,说明沙眼衣原体的种特异抗原决定簇位于各型MOMP上;而与型特异单克隆抗体有反应关系的型特异抗原决定簇只分布于鼠肺炎株的MOMP上。并用放射免疫法探讨了种特异单克隆抗体与鹦鹉热衣原体脑膜肺炎株外膜主蛋白抗原间的相互关系。     

曼氏血吸虫卵可溶性抗原基因克隆的研究

本文报导以曼氏血吸虫(Schistosoma mansoni)成虫mRNA为模板,通过逆转录酶促法合成cDNA。将虫源cDNA插入载体λgtll噬菌体基团组中编码β-半乳糖苷酶的LacZ基因区形成重组DNA,然后导入受体细胞E.coli KM392pmc9(lon~+)建立基因库,并从中筛选出若干株血吸虫抗原基因克隆。一株被兔抗虫卵可溶性抗原高免血清检获的基因克隆,其虫源基因片段经分子杂交证明系单拷贝基因。重组基因在溶原菌株中所表达的融合蛋白通过免疫印迹转移试验(EITB)证明与相关抗体具有很强的结合能力。DNA序列分析揭示该基因编码的多肽系由239个残基组成,其分子量理论推导值为28.7KD,化学性质呈强硷性和强亲水性。引人注意的是此多肽涟中含有一个由7个“精氨酸—甘氨酸”重复组成的结构独特片段。     

副猪嗜血杆菌广东分离株外膜蛋白P5基因的克隆及蛋白结构预测

根据GenBank上发表的副猪嗜血杆菌(HPS)外膜蛋白基因的核苷酸序列设计并合成1对特异性引物,从广东省HPS分离株外膜蛋白P5基因中扩增出与预期设计的1116bp大小相符的片段,将扩增产物连接到pMD18-T载体上,进行序列测定和分析。结果表明,克隆出HPS广东分离株的目的基因,核苷酸长为1116bp,共编码371个氨基酸,与已发表的HPS(SH0165)外膜蛋白基因核苷酸序列同源性100%,氨基酸同源性100%。生物学预测分析结果显示,HPS外膜蛋白是一种混合型结构蛋白,含有α-螺旋、β-折叠、β-转角和无规则卷曲,其β-转角和无规则卷曲区域可能形成抗原表位;其N端含有1个信号肽,最佳切割位点在21～22个氨基酸;有15个抗原决定簇;无跨膜区;同源建模分析,未见相似三维结构。     

猪流产鹦鹉衣原体的抗原结构及抗原性和免疫原性分析

本试验用ＳＤＳ－ＰＡＧＥ技术对分离自青海、湖北、广西省流产猪的鹦鹉衣原体抗原结构进行分析，发现这些菌株的抗原结构图谱相似，即共同含有主外膜蛋白ＭＯＭＰ和其他一些主多肽。并以Ｄ１３株免疫兔子制备的抗血清作为探针，用免疫印迹试验检查其与各株鹦鹉衣原体抗原分子的反应关系。证实Ｄ１３株的主要抗原３８ＫＤ（ＭＯＭＰ）、６０ＫＤ、８０ＫＤ和１１０ＫＤ具有抗原性，它们可刺激机体产生抗体免疫应答。该抗血清不仅可与     

羊流产嗜性衣原体MOMP基因的克隆及序列分析

为探讨羊流产嗜性衣原体MOMP蛋白的生物学功能、揭示其在流产衣原体病诊断和防治中的作用。本试验根据Gen Bank公布的流产嗜性衣原体主要外膜蛋白(MOMP)基因序列,设计并合成4条特异性引物,以羊流产嗜性衣原体青海分离株感染的鸡胚卵黄囊中提取的衣原体基因组为模板,应用套式PCR扩增到MOMP全基因序列。通过生物信息学软件对该基因的结构及其推导的氨基酸序列进行了预测分析,同时分析了与其他流产嗜性衣原体的同源性。结果显示,该基因全长1 170 bp,可编码389个氨基酸,蛋白分子量理论值为41.8 ku,理论等电点7.54,编码蛋白理化性质较稳定。其第1位到第19位氨基酸残基为信号肽序列,具有3处N-糖基化位点。含有多个能被其他酶修饰的位点,以无规则卷曲为主,含5个跨膜区,3个亲水性较强的抗原表位。同源性分析表明与其他流产嗜性衣原体分离株的一致性高在93%以上,系统进化分析表明与OCLH196株(序列号为AJ440239)位于同一进化分支。     

多株猪源大肠杆菌结构抗原和分泌抗原的研究

选取6株(300、303、316、325、327和343)猪源大肠杆菌免疫家兔制备高免血清,与6株菌的全菌抗原、菌体(O)抗原、渗透因子及外膜蛋白抗原分别进行交叉凝集试验和琼脂扩散试验。结果表明,大肠杆菌的主要抗原为O抗原,其中300和303 2株菌的抗体与其他4株菌的抗原具有广泛的交叉性。     

猪传染性胃肠炎与猪流行性腹泻病毒抗原成份分析

采用聚乙二醇沉淀法结合蔗糖梯度密度离心法纯化了猪传染性胃肠炎(TGE)和猪流行性腹泻(PED)两种病毒.SDS-PAGE 分析表明,TGEV 有六种蛋白成份,依次为 VP_1(33KD)、VP_2(41KD)、VP_3(58KD)、VP_4(60KD)、VP_5(142KD)和 VP_6(200KD);PEDV亦有六种蛋白成份.为VP_1(33KD)、VP_2(40KD)、VP_3(53KD)、VP_4(62KD)、VP_5(163KD)和VP_6(190KD).两种病毒的结构蛋白间的差异主要表现在 VP_5上.Westen-Blot 分析证明,TGEV 与 PEDV 在VP_2和 VP_4处存在抗原活性交叉反应,其它结构蛋白则无,VP_1和 VP_2为基质蛋白,VP_3和 VP_4为核蛋白,VP_5和 VP_6为纤突蛋白,病毒纤突蛋白可诱生中和抗体.因此认为,TGEV 和 PEDV 在 VP_5结构蛋白上表现出来的差异很可能是导致两种病毒间无血清学交叉反应的分子基础.两种病毒在 VP_2和 VP_4处有抗原活性交叉反应,这可能由于两种病毒的基质蛋白和核蛋白在生物进化中有一定亲缘关系。     

鹦鹉热衣原体亚组分和亚单位制剂的保护力

用小鼠毒性效力试验检测了鹦鹉热衣原体（Ｂａｋｅｒ株）亚组分及亚单位疫苗的保护力。以衣原体的原生小体（ＥＢ）、外膜复合膜（ＣＯＭＣ）、外膜主蛋白（ＭＯＭＰ）、脂多糖（ＬＰＳ）及ＭＯＭＰ＋ＬＰＳ混合物作为制苗抗原。按以前描述的对沙眼衣原体离心和去污提取法制备鹦鹉热衣原体的ＥＢ和ＣＯＭＣ。通过将衣原体感染组织培养液做聚丙烯酰胺凝胶电流和从凝胶上洗脱，分离和纯化ＭＯＭＰ和ＬＰＳ。从ＥＢ用层析法提取的ＭＯＭ     

动物布鲁氏茵病的流行情况及防控措施

1 病原学特点 布鲁氏菌是自然疫源性疾病，抗原结构复杂，主要成分是脂多糖和高度特异的外膜蛋白。脂多糖包含多糖0抗原、多糖核和类脂。外膜是细菌细胞与外环境分界的屏障，具有高特异性结构，其成分是磷脂中嵌入1组特异蛋白。     

Antigen heterogeneity among Mycoplasma mycoides subsp. mycoides SC isolates: discrimination of major surface proteins

The protein and antigen profiles of 60 isolates, strains and the type strain PG1 of Mycoplasma mycoides subsp. mycoides SC were compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblot analysis. Analysis using contagious bovine pleuropneumonia antisera and hyperimmune rabbit sera against several representative strains revealed some differences in protein profiles and variability in antigens among strains from different geographic regions. The most common antigenic bands had the molecular masses of 110, 95, 80, 69, 62, 60, 48, 44, 39 and 38 kDa. There were differences among European strains, where a larger group coming from Italy lacked the p98 antigen, thus, with one exception, distinguishing the Italian strains from Portuguese, French and Spanish strains. African, Australian and PG1 strains showed heterogenic profiles, with quantitative differences and in a few strains some antigenic bands were absent. The group constituting African, Australian and PG1 strains was characterised by the presence of 71.5/70 kDa antigens, which were not detected in European strains. Mycoplasma mycoides subsp. mycoides SC membrane proteins were characterised by Triton X-114 partitioning and p110, p98, p95, p62/60 and p48 were identified as immunogenic antigens. The simultaneous presence of these five antigens was common to all the sera examined and, therefore, indicates the diagnostic potential of immunoblotting. Most immunodominant antigens are surface-exposed proteins as determined by the trypsin treatment.     

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     

奶牛Y精子膜蛋白的提取与分析

奶牛Y精子膜蛋白的提取与鉴定为深入研究Y精子特异膜蛋白在受精过程中的作用，开发经济、安全、高效的奶牛性别控制方法具有重要意义。本研究采用超声波破碎法将精子膜与精子分离，分离后的精子膜粗品采用蔗糖密度梯度离心法进行纯化。纯化的精子膜经膜蛋白提取液（去污剂）提取，透析浓缩。经变性聚丙烯酰胺凝胶电泳（SDS—PAGE）银盐染色，成功地提取到膜蛋白。BIO—RAD凝胶成像分析仪分析得知Y精子膜蛋白至少有10种，分子量范围为7．94KD-166．65KD，其中尤以15KD的蛋白含量最多。     

Biological properties and genetic analysis of the ompA locus in chlamydiae isolated from swine.

Eight strains of Chlamydia psittaci isolated from swine with pneumonia, pleuritis, pericarditis, and enteritis were characterized through analysis of the major outer membrane protein gene ompA by a two-step polymerase chain reaction, by their interactions with cells in culture, and by the morphologic features and ultrastructure of intracellular inclusions. Amplified chlamydial ompA DNA fragments were differentiated by restriction endonuclease digestion. Chlamydial isolates were separated into 2 types on the basis of ompA restriction fragment length polymorphism. Strains of type L71 had finely granular inclusions, whereas those of type 1710S contained pleomorphic reticulate bodies (RB) in the inclusions, which are characteristic of aberrant chlamydial developmental forms. Chlamydial types L71 and 1710S required centrifuge-assisted inoculation for efficient infection of cell cultures. Cultivation in cell culture medium containing cycloheximide increased the numbers of chlamydial inclusions about 1.5-fold. These strains formed few elementary bodies in yolk sac cells of chicken embryos. Ultrastructurally, unique doublet RB were observed, particularly in strains of the ompA type L71. These doublets consisted of 2 RB, bounded by a cytoplasmic membrane, contained within a common cell wall and an extended periplasmic space. Ultrastructural examination of strains of the ompA type 1710S confirmed the aberrant chlamydial developmental forms, but evidence of viral infection of the RB as a cause of these aberrant forms was not found. The strain S45 isolated from intestinal sites of swine was a trachoma restriction fragment length polymorphism type. With the mouse biotype, it represented the second isolate from animals of Chlamydia trachomatis.     

Genotype and sex effects on the relationship between energy intake and protein deposition in growing pigs

Seventy-two crossbred (Large White X Landrace) pigs were used in a 3 X 7 factorial experiment to investigate the response of two strains of boars (strains A and B) and of castrated male pigs (strain B) to seven levels of intake of a single diet (ranging from 5.3 Mcal digestible energy [DE]/d to ad libitum) between 45 and 90 kg live weight. All aspects of growth performance and body composition were affected to different degrees by both strain and sex. At all levels of energy intake strain A boars grew faster, had a lower feed to gain ratio and contained less fat and more water in the empty body than strain B boars, which in turn exhibited faster live weight gain and more efficient and leaner growth than castrated males. The magnitude of the differences in growth performance between strain A and strain B boars and castrates increased with increased energy intake above 7.88 Mcal DE/d, and these differences were associated with concomitant strain differences in their respective capacity for protein growth and in the relationship between energy intake and protein deposition. For strain A boars the rate of protein deposition increased linearly from 92 to 188 g/d with increased energy intake from 5.3 Mcal DE/d to ad libitum. For strain B boars and castrates the rate of protein deposition increased linearly with increased energy intake up to 7.88 Mcal DE/d, but thereafter it remained constant at 128 and 85 g/d, respectively. For castrates protein deposition was depressed (P less than .01) when the diet was offered ad libitum. Strain A boars had a higher energy requirement for maintenance (3.55 Mcal DE/d) than strain B boars (2.77 Mcal DE/d) or castrates (2.60 Mcal DE/d). Strain A boars also contained less protein and more water in the fat free empty body than the other two pig types.     

大肠杆菌耐药性抑制剂作用机制的初步研究

20株人源和动物源性大肠杆菌在体外经黄连提取物(命名为耐药性抑制剂IT2)作用后,对氟喹诺酮类抗菌药、庆大霉素和多西环素的耐药性明显被抑制.选取经抑制剂IT2作用后耐药性变化显著的5对菌株进行质粒提取和电泳检测,发现J0、H004、J3、J2 等4株菌作用前均有质粒带,与其相对应的耐药性抑制剂作用后的菌株ITJ0、ITH004、ITJ3、ITJ2质粒带全部消失,只有菌株J1经抑制剂IT2作用前后质粒带无变化.提取外膜蛋白进行SDS-PAGE电泳分析,菌株J1、J3、J0缺少OmpF、OmpA, J2仅缺少OmpA,经耐药性抑制剂作用后,J1、J3、J0的OmpF均由缺失恢复为表达,OmpC的表达量呈显著增加,H004没有变化.结果表明耐药性抑制剂IT2对大肠杆菌的部分耐药性具有良好的抑制作用,其作用与阻碍耐药质粒拷贝、恢复外膜通道蛋白正常表达关系密切.     

Variations in the virulence of strains of Chlamydia psittaci for pregnant ewes

Invasive and non-invasive strains of Chlamydia psittaci isolated from faeces of clinically healthy ewes and from vaginal swabs of ewes which had aborted were injected intravenously or intradermally into pregnant ewes. The results were studied by recording the ewes' thermal and serological responses, lambing performance and the excretion of chlamydia from the vagina. The differences between the effects of different invasive strains were greater after intradermal inoculation than after intravenous inoculation. After intradermal inoculation non-invasive strains did not disturb pregnancy (11 of 13 ewes lambed normally) whereas invasive strains induced abortion in 23 of 25 ewes, 24 of which excreted chlamydia in vaginal secretions.     

Comparative study of various Chlamydia strains--preliminary study (author's transl)]

The chlamydia order comprises two species (Chlamydia trachomatis and Chlamydia psittaci) which are the only ones which can be rigorously differentiated with experimental criteria. However, the clinical study of chlamydia demonstrates the existence of various syndromes due to pathogenic agents. Some observations also seem to indicate the possibility of antigenic differences between isolated strains, during abortions, among small ruminants.We have entered upon a comparative study of various Chlamydia psittaci strains in order to look for objective criteria of differentiation of the strains extracted from small ruminants from those which were isolated from other animal species. Chlamydia taken from samples of ovine and caprine origin by direct isolation on embryonated egg or on cellular cultures were also compared by the following methods: seroneutralization on embryonated eggs and on cell cultures, characteristics of the plaques on cell cultures, crossed-immunofluorescence, toxic effects, lethal action on the foetus of the pregnant mouse, crossed-immunoelectrophoresis by agar gel (simple or double quantitative diffusion), electrophoresis by polyacrylamide gel.The use of those numerous techniques has enabled us to observe significant differences between those strains. However, it is not yet possible to propose a classification defining several groups. The specificity of these differences, especially between ovine and caprine strains, should become clear through the studies now in progress: in particular through the method using the interference of specific antigens compared on the one hand by serioimmunologic methods and, on the other hand, by tests setting an immunity to cellular mediation into action.     

Proteome-determined type-specific proteins of Mycobacterium avium subspecies paratuberculosis

Mycobacterium avium subspecies paratuberculosis (M. a. paratuberculosis) is a pathogen of ruminants, causing paratuberculosis (characterized by severe emaciation). The disease is endemic in many countries including the UK and places a severe economic burden on the global livestock industry. Two types of M. a. paratuberculosis can be classified by pulsed-field electrophoresis (I/III and II), which are phenotypically distinct and appear to have different host preferences. Proteomes of Type I and Type II M. a. paratuberculosis were analyzed by 2-D gel electrophoresis to determine if any significant differences existed between the subtypes. Seven different strains of Type I and 18 strains of Type II were analyzed and compared to detect type-specific differences. These 'type-specific' differences existed regardless of growth phase and were also exhibited in cells isolated directly from pathogenic lesions. Twenty-three spots predominated on the Type I profile, from which 17 proteins were identified. Twenty-one spots predominated on the Type II profile, from which 16 proteins were identified. None of the proteins identified as differentially represented on the profiles of Type I or Type II corresponded to open reading frames of the defining genomic regions as previously described for the Type I (sheep) and Type II (cattle). Sequence polymorphisms existing in Type I and II strains were identified in some open reading frames or regulatory regions of genes that correspond to proteins expressed in a type-specific fashion. The consequence of these is discussed in relation to protein expression and their impact on the type phenotype is discussed.     

禽腺病毒贵州分离株ＨＳ—１的核酸酶切分析与蛋白抗原鉴定

本研究利用１０％ＳＤＳ－ＰＡＧＥ及Ｗestern-Blotting技术鉴定３株不同的减蛋综合征病毒（贵州株ＨＳ－１、南京分离毒ＧＣ２、国际标准毒ＡＶ－１２７）的蛋白抗原性，结果表明，３株毒的蛋白多肽分子量均在１０６－１２kＤ范围,一各条多肽的组成上略有差异；它们都具有两条相同的免疫原性多肽，分子量分别为１０６和１００ＫＤ。同时，运用ＥcoRⅠ,HindⅢ，ＰｓｔⅠ和ＳmaⅠ等５种限制性内切酶进行了酶切分析，证实３株ＥＤＳ病毒用ＰstⅠ和ＳmsⅠ酶切的片段大小及长度略有不同，而其它３种酶的酶切图谱完全相同。