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1.
猪链球菌2型作为一种人兽共患病病原,日益受到关注。而溶血素是其分泌的外毒素,是公认的重要毒力因子之一,具有良好的免疫原性。本试验通过PCR方法获得猪链球菌2型野生型溶血素基因sly和463、464双点突变的溶血素突变体基因slym。将sly和slym克隆至表达载体,构建了2个重组载体并在大肠杆菌中表达了野生型溶血素rSLY和突变型溶血素rSLYm。经过蛋白杂交试验,证明表达的rSLY和rSLYm与提取的猪链球菌的天然野生型SLY分子量完全一致。以提取的野生型SLY为对照,通过溶血试验证明,突变型溶血素rSLYm失去了溶血活性;通过接种PK15、RK13、SUVEC细胞单层,证明突变型溶血素rSLYm失去细胞毒性;通过小鼠试验,证明突变型SLYm对小鼠没有毒力。本试验通过溶血试验、细胞接种和小鼠实验,证明双点突变灭活了野生型溶血素的溶血活性、细胞毒性和小鼠毒力。该溶血素突变体经免疫实验证实后,可作为猪链球菌2型亚单位疫苗的候选抗原。  相似文献   

2.
根据已发表的嗜水气单胞菌（Aeromonas hydrophila,Ah）溶血素A基因序列设计一对特异性引物,以TPS30菌株基因组为模板,通过PCR扩增溶血素A基因,经T-A克隆、序列测定和分析,结果表明,该基因包含1482 bp碱基,编码494个氨基酸。将溶血素A定向克隆至表达载体pET32a（＋）中,构建重组表达质粒pET-THA,在IPTG诱导下成功获得重组表达蛋白His-GroEL,大小为68 kDa。对该质粒在大肠杆菌BL21（DE3）中的表达特性分析表明,最佳的诱导表达条件为：在0.1 mmol/L的IPTG浓度下,21℃诱导表达3 h。表达的溶血素A蛋白纯化后免疫小鼠,经Western blot检测,表明该蛋白具有免疫原性。对纯化的溶血素A蛋白复性后,进行溶血实验,结果表明该蛋白具有溶血活性。通过本实验的研究,为进一步研究嗜水气单胞菌溶血素的生物学功能奠定了基础。  相似文献   

3.
为了探讨猪链球菌2型(Streptococcus suis serotype 2,SS2)的Ⅲ型溶血素是否具有溶血活性以及Ⅲ型溶血素在SS2致病过程中的作用,本研究利用同源重组基因敲除法成功构建了SS205ZY的Ⅲ型溶血素(slyrp)基因缺失突变菌株△slyrp及双基因缺失突变菌株△sly/△slyrp,并比较了野生菌株和基因缺失突变菌株的溶血能力以及对小鼠的致病力.结果表明,slyrp基因敲除后可导致SS2裂解红细胞的能力有所下降,而双基因缺失突变菌株△sly/△slyrp的溶血能力完全丧失;slyrp基因敲除后对小鼠的致病力没有影响.结果提示猪链球菌2型Ⅲ型溶血素具有一定的溶血能力,该Ⅲ型溶血素在SS2感染过程中,对溶血素(sly)起协同作用,不是SS2主要的毒力相关基因.  相似文献   

4.
为研究温和气单胞菌(A.sobria)溶血素基因的特性,本研究根据GenBank登录的气单胞菌属溶血素基因序列设计一对引物,经PCR扩增得到大小约1.5kb的A.sobria RC-07-KA株溶血基因片段,将该片段克隆到pGEM-T载体,测序结果显示:溶血素基因片段大小为1 467 bp,编码487个氨基酸残基,遗传进化分析结果表明该基因与气单胞菌属中的A.hydrophila菌株Sb (AY611033)、NLEPA-1607 (AF410466)、AEF (HM853019),A.sobria菌株357 (AY157998)、人源分离株(EF620533)和A.salmonicida菌株17-2 (X65048)的溶血素基因亲缘关系较近,同源性大于95％,而与其他菌株的同源性较低.通过构建溶血素重组表达质粒pET-HIy,诱导表达并通过western blot鉴定表达蛋白,结果显示重组菌能高效表达重组溶血素,而且纯化的重组溶血素具有溶解鲤鱼红细胞的活性.为该菌进一步深入研究奠定了基础.  相似文献   

5.
本实验从鸡肠道分离出50株菌种,通过对菌种菌落培养特性、形态观察、生理生化实验,初步鉴定出8株为芽孢杆菌。通过产蛋白菌株的筛选试验筛选出1株能够分泌蛋白的芽孢杆菌,该菌株可望成为外源蛋白高效分泌表达的宿主。  相似文献   

6.
流产布鲁氏菌疫苗候选株RB6生物学特性研究   总被引：1，自引：0，他引：1  
为了开发布鲁氏菌病新型标记疫苗,本研究对流产布鲁氏菌基因缺失株RB6的培养特性、染色特性、凝集特性、稳定性及小鼠体内毒力和免疫保护力进行了系统鉴定,旨在阐明该菌株具备的生物学特性。通过对亲本菌株和RB6的比较,发现固体培养基上RB6菌株单菌落可被结晶紫染成紫色,RB6菌株液体培养物可与0.1%吖啶黄染料以及抗布鲁氏菌粗糙型抗体发生凝集反应,证明该菌株为粗糙型。将RB6菌株在体外连续传代培养20次和牛体内连续5次继代,检测结果证明其表型未发生变化,说明该菌株遗传稳定性良好。通过小鼠体内试验发现该菌株毒力显著降低,并对流产布鲁氏菌强毒菌株2308攻毒的免疫保护力与现有疫苗A19接近。本研究结果表明,流产布鲁氏菌基因缺失株RB6为粗糙型菌株,毒力较低、安全性高、遗传性状稳定,并具有良好的免疫保护效力,有望开发成为动物布鲁氏菌病粗糙型标记疫苗。  相似文献   

7.
为鉴定胸膜肺炎放线杆菌（Actinobacillus pleuropneumoniae，APP）ApxIA毒素蛋白N端疏水区和C端Ca^2＋结合区的免疫原性，参照apxIA基因序列（D16582）设计4条引物，用于扩增APP血清10型参考菌株（D13039）基因组DNA中长约3．2kb的apxIA基因及其N端（1．4kb）和C端（1．8kb）基因片段，经克隆测序后分别插入原核表达载体pET-32a中进行表达，表达的融合蛋白大小分别约为125Ku、65Ku和80Ku。表达产物免疫小鼠后的攻毒保护试验结果显示，ApxIA表达蛋白对APP血清10型（D13039）攻毒可提供完全保护，而对APP血清1型（4074）攻毒仅能提供部分保护，与提纯的Apx1毒素蛋白免疫保护效果相当；ApxIA-N端表达蛋白免疫保护活性显著高于ApxIA-C端表达蛋白，提示ApxIA蛋白免疫活性位点多位于N端，在Apx1毒素蛋白的保护性抗原活性中发挥更重要作用。  相似文献   

8.
猪传染性胸膜肺炎（Porcine infectious pleuropneumonia）是由胸膜肺炎放线杆菌（Actinobacillus pleuropneumoniae，APP）引起的猪的一种呼吸道疾病。本实验以APP血清7型的一株现地分离株L25—4株为研究对象，对其分泌的ApxⅡ毒素的结构基因apxⅡA进行了全基因克隆和测序，并利用原核表达载体pGEX-6P-1在E．coli中进行了原核表达。表达产物以包涵体形式存在，包涵体蛋白经过洗涤后作为抗原用于Western—blotting免疫印迹实验，结果表明重组的ApxⅡA蛋白具有良好的免疫原性。  相似文献   

9.
本试验从鸡肠道中分离出菌种50株,通过对菌种菌落培养特性、形态观察、生理生化实验,初步鉴定出8株为芽胞杆菌。经产蛋白菌株的筛选试验筛选出1株能够分泌蛋白的芽胞杆菌,该菌株可望成为外源蛋白高效分泌表达的宿主。  相似文献   

10.
从西藏地区腹泻死亡牦牛中分离出一株肠毒素型大肠杆菌并对其某些生物学特性进行了研究。结果表明，该菌在形态特`征培养特性和生化特性方面与大肠杆菌基本一致。血清学试验表明，该菌株O抗原属O148，K88、K99、987P单因子血清均不能凝集本菌；该菌不产生溶血素；对绵羊、豚鼠、马、鸡的红细胞表现强凝集，而K88、K99、987P抗血清均不能抑制其它对绵羊、豚鼠、马、鸡红细胞的凝集；该菌株在营养肉汤中经37℃，48小时培养表达菌毛；肌肉接种兔、腹腔接种小鼠均具有高致病性；乳鼠胃内投报试验和兔回肠结扎试验证明，该菌能产生热稳定肠毒素和热敏性肠毒素；分离菌株对恩诺沙星、环丙沙星、阿莫西林、羧苄青霉素等高度敏感，而对链霉素、四环素、土霉素等表现耐药性。  相似文献   

11.
Ten strains of H. pleuropneumoniae isolated from 10 herd outbreaks of pleuropneumonia were studied by means of the slide agglutination test, the indirect haemaggluitiniation (IHA) test and by gel diffusion. The strains were antigenically homogeneous and serologically distinct from serotypes 1 through 8. It is therefore proposed to refer these strains to a new serotype: serotype 9, with strain CVJ 13261 as the type strain.In addition to the serotype-specific capsular antigens, capsular antigen of serotype 1 (strain 4074) could be demonstrated in the 10 strains by means of gel diffusion analyses.In cross protection studies it was shown that the antigenic determinants shared by serotypes 9 and 1 were unable to yield a sufficient protection against disease. Thus, parenteral immunization with a killed 6-h culture of serotype 9 did not afford an acceptable protection against challenge with serotype 1 since only 3 of the 5 vaccinates were protected. The reverse experiment showed that parenteral immunization with serotype 1 only protected 1 out of 4 vaccinates.  相似文献   

12.
An efficient, single-step method for purification of the 110-kilodalton (kDa) hemolysin of Actinobacillus pleuropneumoniae was developed. An immunoaffinity column was made by cross-linking murine monoclonal antibody 8C2 to the 110-kDa hemolysin of A pleuropneumoniae strain J45 serotype 5 to protein A-agarose beads. Purified hemolysin with high hemolytic activity was obtained after washing the column with phosphate-buffered saline solution, and eluting the hemolysin with 50 mM diethylamine, pH 11.0. The same column was also used to purify the hemolysin from A pleuropneumoniae strain 4074 serotype 1. The purification procedure could be completed within 5 hours, and almost 50% of the total hemolytic activity and hemolysin protein was recovered in pure form.  相似文献   

13.
【目的】根据鸭疫里氏杆菌(Riemerella anatipestifer,RA)血清1型、2型贵州流行株制备二价灭活疫苗,为鸭疫里氏杆菌病的防控及疫苗研制提供研究资料。【方法】以血清1型RA(RA-G06株)、血清2型RA(RA-HS01株)地方流行株为菌种,通过涂板法测定菌株生长曲线,利用改良寇氏法计算菌株对鸭的半数致死量(median lethal dose, LD50),将2株菌培养至终浓度为1×1010 CFU/mL后等比例混合,以卡波姆为佐剂制备二价灭活疫苗,经疫苗质量检验后进行雏鸭免疫试验;通过检测免疫鸭血清中特异性抗体水平和攻毒保护试验评价疫苗的保护率,对攻毒试验鸭心脏、肝脏、脾脏和脑组织进行组织病理学观察。【结果】RA-G06株和RA-HS01株均在培养12 h时到达峰值,活菌数分别为2.1×1011和3.3×1011 CFU/mL,LD50分别为1.44×1010和2.63×108 CFU/mL;制备的疫苗安全性良...  相似文献   

14.
Streptococcus suis diseases in pigs, most importantly meningitis, are worldwide responsible for major economic losses in the pig industry. About one fourth of invasive S. suis diseases are caused by S. suis serotype 9 strains in Europe. However, little is known about serotype 9 since most studies were performed with serotype 2. The objective of this study was to determine the immunogenicity and protective efficacy of a serotype 9 bacterin in piglets. Challenge was conducted with a reference serotype 9 strain, belonging to the same clonal complex but to a different sequence type as the bacterin strain. The bacterin induced protection against mortality but not morbidity. Eleven days post infection, 3 of 7 vaccinated survivors were not fully convalescent and had not eliminated the challenge strain from inner organs completely. In accordance with the clinical findings, the majority of piglets showed fibrinous-suppurative lesions in at least one inner organ or tissue. In contrast to the placebo group such lesions were not detected in one third of bacterin-vaccinated piglets. Determination of specific serum IgG titers revealed that the bacterin elicited seroconversion against muramidase-released protein and basic membrane lipoprotein. Furthermore, vaccination was associated with induction of opsonizing antibodies against the serotype 9 challenge strain. However, titers of opsonizing antibodies were rather low in comparison to those found in our previous serotype 2 vaccination trial. Piglets developed substantially higher titers of opsonizing antibodies after challenge. Opsonizing antibodies were absorbable with the serotype 9 challenge strain but not with an unencapsulated isogenic mutant of a serotype 2 strain indicating their specificity. The results indicate that a serotype 9 bacterin is less protective than a serotype 2 bacterin, most likely due to inducing only low titers of opsonizing antibodies. This might contribute to emergence of serotype 9 strains, in particular strains of this clonal complex, in Europe.  相似文献   

15.
Cross-protection induced by primary infection with Abortusovis and Gallinarum was examined against challenge injection with these Salmonella serotypes as well as with Dublin and Choleraesuis, the other virulent serotypes. Abortusovis induced efficient protection against the other Salmonella. Gallinarum was ineffective against Choleraesuis. Even with low multiplication in mice, the Gallinarum J91 strain induced a weak but significant protection against Dublin (same O group serotype). The antibodies in the blood of mice were tested with ELISA specific for the Salmonella antigens used to prime or to challenge animals. The Gallinarum J91 strain was detected to be more antigenic in ELISA than the other Salmonella antigens. It is difficult to conclude on a correlation between IgM or IgG antibodies and induction of protection, because of the variability in immune response according to the different serotype used. Nevertheless, the negative linkage between a number of bacteria in the spleen of mice challenged with Gallinarum and Dublin, and the level of IgM and IgG antibodies specific for the challenging serotype, showed that humoral immune response could be one element of cross-protection, mainly by the immune response against the same O serotype.  相似文献   

16.
The immunogenic and protective potentials of an outer membrane-enriched fraction (OM) from a serotype 5 strain of Actinobacillus (Haemophilus) pleuropneumoniae (APP) and the same OM degraded with proteinase K or periodate were evaluated in swine. Groups of pigs were vaccinated with two doses of OM, proteinase K-treated OM (P-OM), periodate-treated OM (PI-OM), or placebo vaccine and challenged intranasally with the homologous strain of APP. Results from triplicate experiments indicated that proteinase K treatment of OM resulted in an improved efficacy. This improved efficacy of P-OM vaccine over untreated OM vaccine was evidenced not only by less severe lung lesions in P-OM vaccinated pigs but also by significant reduction (P less than 0.05) in the number of P-OM vaccinated pigs which developed lung lesions upon challenge with APP. Assessment of sera from vaccinated animals by immunoblotting, complement fixation test, or ELISA indicated that the immunogenicity of some but not all protein or carbohydrate components were reduced (or eliminated) by proteinase K and periodate treatments respectively.  相似文献   

17.
A trial was performed in a swine research facility to ascertain the protection provided by a polyvalent Actinobacillus pleuropneumoniae (APP) bacterin containing serotypes 1, 3, 5 and 9. The test animals consisted of 60, eight-week-old, piglets, which were randomly divided into four main groups. The four main groups were further divided into three sub-groups (I, II, III) of five pigs each. Subgroup I was vaccinated intramuscularly, sub-group II was vaccinated subcutaneously, and sub-group III served as the unvaccinated control group. Each main group was challenged with a single APP serotype (1, 3, 5 or 9). Criteria for evaluation of the bacterin efficacy were mortality, lung lesions, pleural adhesions, and isolation of APP from tonsil or lung. Significant effects of vaccination over nonvaccination were reduced mortality, lung lesions, pleural adhesions, and isolations of APP from tonsil and lung. There were no significant differences between the intramuscular and subcutaneous routes of vaccination. It was concluded that the four-way APP bacterin used in this study provided satisfactory protection against homologous challenge. Evidence of protection was lower mortality and lung lesions and increased daily weight gains in vaccinates as compared with controls.  相似文献   

18.
Two experiments were done to further define cell-free culture filtrate (CCF) from Pasteurella multocida and its endotoxin content in protecting turkeys against challenge. In the first experiment, the greater-than-30,000-molecular-weight fraction of P. multocida strain R44/6 (serotype 3/4/9/12) CCF was used in 10-fold dilutions given by air-sac inoculation or aerosol to vaccinate turkeys, which were subsequently challenged with either homologous (P-1059, serotype 3) or heterologous (X-73, serotype 1) strains. Endotoxin content of the CCF fraction was high. Compared with positive controls given either live Clemson University vaccine or a commercial bacterin, homologous protection was provided by undiluted CCF and 1:10 dilutions of CCF, but there was no heterologous protection. In the second experiment, CCF of strain R44/6 in regular and iron-limiting media and CCF of strain FC127B (serotype 1/4) were used alone or in combination to vaccinate turkeys, which were challenged as in the first experiment. Homologous but not heterologous protection occurred, even though growth of strain R44/6 in iron-limiting media reduced endotoxin content of CCF by approximately 93%. These results indicate that endotoxin levels of less than 10% but greater than 1% of those in CCF from regular media are sufficient to induce protection in turkeys against homologous challenge but that CCF from either regular or iron-limiting medium does not provide protection against heterologous challenge.  相似文献   

19.
Vaccination of pigs with a killed culture of A. pleuropneumoniae serotype 5, strain K17 (subtype a) afforded a high degree of protection against challenge with strains L20 and T928 (subtype b). The reverse experiment showed that strain L20 gave good protection against challenge with strain K17 whereas strain T928 did not afford an acceptable protection against challenge with this strain.The considerable cross immunity shown to exist between strains K17 and L20 indicates a high degree of homogeneity of the antigenic determinants of the two strains involved in induction of protective immunity and suggest that antibodies to capsular subtype specific determinants may not play a significant role in the specific defence against A. pleuropneumoniae strains belonging to serotype 5. The finding that a vaccine prepared from strain T928 did not afford an acceptable protection against challenge with strain K17 indicates a variable expression among serotype 5 strains of the antigenic determinants which induce protective immunity against A. pleuropneumoniae infection.  相似文献   

20.
【目的】 研究针对新城疫病毒(Newcastle disease virus, NDV)和禽腺病毒(Fowl adenovirus, FAdV)的耐热基因工程疫苗。【方法】 利用反向遗传学操作技术将NDV耐热株的HN基因替换到LaSota疫苗株上, 再将禽腺病毒血清4型(Fowl adenovirus serotype 4, FAdV-4)的Fiber2基因插入到其基因组上, 构建表达Fiber2蛋白的重组耐热NDV质粒pTS-HN-Fiber2。通过病毒拯救技术拯救重组NDV rTS-HN-Fiber2, 并测定其生物学特性和作为疫苗候选株的免疫原性和攻毒保护性。【结果】 rTS-HN-Fiber2的鸡胚平均致死时间>168 h, 且脑内接种致病指数为0, 属于弱毒的范畴; 在细胞上的生长曲线结果表明, rTS-HN-Fiber2与亲本LaSota株有相似的生长曲线, 但最终的生长滴度略低于LaSota株; rTS-HN-Fiber2在56 ℃处理15 min后, 病毒滴度下降约103 TCID50/mL, 而LaSota株56 ℃处理5 min几乎无感染性; 间接免疫荧光试验结果表明, rTS-HN-Fiber2能表达Fiber2蛋白。免疫和攻毒试验结果显示, rTS-HN-Fiber2能产生NDV抗体, 且能显著提高雏鸡在FAdV-4强毒下的存活率, 减轻FAdV-4强毒引起的组织病变, 降低组织中的病毒载量。【结论】 本研究成功构建了表达FAdV-4 Fiber2蛋白的重组耐热NDV, 该病毒保持了亲本LaSota株的弱毒生物学特性, 但热稳定性有显著提升; 重组NDV免疫雏鸡可产生针对NDV和FAdV-4强毒的保护, 该重组NDV可作为开发针对FAdV-4和NDV二联基因工程疫苗的候选病毒株。  相似文献   

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