禽波氏杆菌OMP标记抗体检测方法的建立及相关毒素生物学特性的检测
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摘要
禽波氏杆菌病是由禽波氏杆菌(Bordetella avium)引起家禽的一种高度接触性传染病。我国最早是由朱瑞良等在1990年初发现的,该病主要引起鸡胚死亡、孵化率降低和雏鸡急性死亡及成年鸡的眼炎等。据报道该病在不同地区的感染率为10%～50%,患病鸡群生长迟缓,饲料报酬低,给养鸡业造成了较大的经济损失。
近年来,细菌细胞膜中的一种重要组成成分外膜蛋白(outer-membrane-protein,OMP)的免疫作用越来越受到科技人员的关注,并进行了研究。实验证明,OMP具有良好的免疫原性,不仅可刺激体液免疫,而且也可刺激细胞免疫作用。因此本试验验提取了禽波氏杆菌的OMP,制备了兔抗禽波氏杆菌OMP高免血清,在此基础上建立起标记抗体检测方法,为快速检测禽波氏杆菌病原或抗体提供了手段,为禽波氏杆菌流行病学调查、临床诊断和快速诊断试剂盒的研制奠定了基础。
禽波氏杆菌的致病机理与其本身所含的一些毒力因子密切相关,据推测这些毒力因子可能在禽波氏杆菌的病理发生和免疫方面产生重要的作用。基于上述目的本课题制备了禽波氏杆菌内毒素精制品,禽波氏杆菌DNT及FHA粗提物,为研究禽波氏杆菌的保护性抗原提供了重要依据。建立的禽波氏杆菌菌株培养物感染离体的SPF鸡胚气管环模型,为禽波氏杆菌其他相关毒素对气管环致病机理的研究奠定了基础。
本课题主要研究内容包括以下几个方面:
试验一、禽波氏杆菌OMP高免血清的制备及其标记抗体检测方法的建立
本试验采用改良的Wooldridge法提取了禽波氏杆菌OMP,通过Bradford方法测定禽波氏杆菌OMP含量为320ug/mL,SDS-PAGE电泳检测禽波氏杆菌P5株OMP含有5种成分,分子量分别为58、47、41、36、24KD;P8株OMP含有6种成分,分子量分别为58、47、41、38、21、16KD。将提取的禽波氏杆菌OMP免疫健康青紫兰家兔,每周免疫1次,共计免疫4次,获得了兔抗禽波氏杆菌OMP高免血清,以此高免血清建立了检测禽波氏杆菌的间接ELISA和IFA的方法,并对其工作条件进行了优化和筛选,试验结果表明间接ELISA抗原、抗血清最佳工作浓度分别为10ug/mL、1:12800,酶标二抗工作浓度为1:5000,最佳包被条件为4℃、24h包被;最佳封闭条件为37℃、1h; IFA兔高免血清稀释度为1:20,FITC标记羊抗兔IgG稀释度为1:10,感作时间为45min时,禽波氏杆菌特异性黄绿色荧光最清晰,非特异性荧光最弱。经临床检测证明该两种方法均具有简便、快速、特异性强的特点,为禽波氏杆菌病的流行病学、血清学调查和临床快速诊断奠定了基础。
试验二、禽波氏杆菌致病性相关毒素的提取及生物学特性检测
本试验对禽波氏杆菌内毒素进行了提纯,并首次粗提了禽波氏杆菌DNT及FHA。用制备的毒素对青紫兰健康家兔、Balb/c小鼠、雏鸡、SPF鸡胚、豚鼠进行动物试验,对禽波氏杆菌的致病性及本身的生物学特性进行研究探讨。同时建立起鸡胚气管环模型,验证气管细胞毒素的存在及其对气管纤毛上皮的致病作用。结果表明:禽波氏杆菌内毒素可使动物呈急性败血症病变。DNT能够致死小白鼠和雏鸡,并对肌肉组织产生损害。FHA只凝集豚鼠红细胞,对家兔和鸡的红细胞不产生凝集作用。禽波氏杆菌P5、P8株分别作10-8.5、10-8.3倍稀释,可使SPF鸡胚气管环接种0.1mL后50%出现气管环纤毛脱落,气管环粘膜出现凹陷、空洞,初步验证了气管细胞毒素的存在及禽波氏杆菌对气管纤毛的损伤机理。对禽波氏杆菌相关毒素的生物学特性的初探为研究禽波氏杆菌的保护性抗原提供了重要依据。
Bordetalla avium disease was a high-contacted infectious disease.In our country,it was discovered by Rui-liang Zhu in 1990.It could arouse that chicken-embryo death;low incubation;chicking acuting death and chicken opHthalmia so on.The infection rate of this disease was 10%～50%,it could bring up illness-chicken grew up lowly, the low redound upon of feedingstuff,and great economy loss.
Recent years, the immunity of outer-membrane-protein of bacteria in cell-wall was paid attention to by science workers.the test testified that OMP with well immunity could stimulate not only humoral immunity and cell immunity. The study indicates that the labelled antibody technique used Bordetella avium OMP is rapid, reliable and sensitive and it has the potential for use as an fast diagnosis for Bordetella avium.
The pathogenesis of Bordetella avium is related to its virulence factors. Presumably, these factors may play an important role in pathology and immunity. In order to solve problems mentioned above, Pure endotoxin、raw dermonecrotic toxin and fibrillae hemagglitinins of Bordetella avium were prepared and chicken embryo tracheal ring model was established for detecting biological characteristics of Bordetella avium.It preliminarity revealed relationship between Bordetella avium toxins and its pathogenicity.
The research divides two parts:
Part One: Preparation of High Titer Immuno Serum and Establishment of detection methods by Bordetella avium OMP with labelled antibody technique
In this study, out membrane proteins of B.avium strains P5 and P8 were prepared by improved Wooldridge method. We could determine contents of Bordetella avium OMP by Bradford, do SDS-PAGE electropHoresis of Bordetella avium OMP and prepare OMP immunity to immunity rabbits for preparation of high titer immuno serum. And then detections of indirect ELISA and indirect immunofluorescence were established by optimization and screening of conditions. The result showed that: the contents of Bordetella avium OMP was 320 ug/mL. The protein molecular weight of P5 and P8 strains is respectively 58、47、41、36、24KD, 58、47、41、38、21、16KD. The detection technique of labelled antibody was simple, rapid and strong specificity. It can be used to study on the epidemiology and serology of bordetella avium. This method laid a foundation of fast diagnosis for bordetella avium.
Part Two: Extraction and detection of biological characteristics of Bordetella avium related toxins
Pure endotoxin、raw dermonecrotic toxin and fibrillae hemagglitinins of Bordetella avium were prepared to used for animal test of rabbits、Balb/c mice、chicks、SPF chicken embryos、guinea pigs. Pathogenicity and biological characteristics of Bordetella avium were researched and explored by animal test. At the same time, chicken embryo tracheal ring model was established to verify the existence of tracheal cytotoxin and its pathogenic effects on tracheal cilia epithelium. The results showed that: endotoxin of Bordetella avium can effectively lead to acute septicemia lesions of animals. DNT caused little white rats、chicks death and muscle tissue damaged. FHA only agglutinated erythrocytes of guinea pigs, it had no agglutination to erythrocytes of rabbit and chicken. When Bordetella avium P5、P8 were diluted 10-8.5、10-8.3 times , abscission and injury of 50% of chicken embryo tracheal rings cilia could be caused by inoculating 0.1mL Bordetella avium.This result preliminarily verified the existence of tracheal cytotoxin and revealed pathogenic mechanism of Bordetella avium on tracheal cilia.The biological characteristics of these toxins played an important role for studing Bordetella avium vaccines.
近年来,细菌细胞膜中的一种重要组成成分外膜蛋白(outer-membrane-protein,OMP)的免疫作用越来越受到科技人员的关注,并进行了研究。实验证明,OMP具有良好的免疫原性,不仅可刺激体液免疫,而且也可刺激细胞免疫作用。因此本试验验提取了禽波氏杆菌的OMP,制备了兔抗禽波氏杆菌OMP高免血清,在此基础上建立起标记抗体检测方法,为快速检测禽波氏杆菌病原或抗体提供了手段,为禽波氏杆菌流行病学调查、临床诊断和快速诊断试剂盒的研制奠定了基础。
禽波氏杆菌的致病机理与其本身所含的一些毒力因子密切相关,据推测这些毒力因子可能在禽波氏杆菌的病理发生和免疫方面产生重要的作用。基于上述目的本课题制备了禽波氏杆菌内毒素精制品,禽波氏杆菌DNT及FHA粗提物,为研究禽波氏杆菌的保护性抗原提供了重要依据。建立的禽波氏杆菌菌株培养物感染离体的SPF鸡胚气管环模型,为禽波氏杆菌其他相关毒素对气管环致病机理的研究奠定了基础。
本课题主要研究内容包括以下几个方面:
试验一、禽波氏杆菌OMP高免血清的制备及其标记抗体检测方法的建立
本试验采用改良的Wooldridge法提取了禽波氏杆菌OMP,通过Bradford方法测定禽波氏杆菌OMP含量为320ug/mL,SDS-PAGE电泳检测禽波氏杆菌P5株OMP含有5种成分,分子量分别为58、47、41、36、24KD;P8株OMP含有6种成分,分子量分别为58、47、41、38、21、16KD。将提取的禽波氏杆菌OMP免疫健康青紫兰家兔,每周免疫1次,共计免疫4次,获得了兔抗禽波氏杆菌OMP高免血清,以此高免血清建立了检测禽波氏杆菌的间接ELISA和IFA的方法,并对其工作条件进行了优化和筛选,试验结果表明间接ELISA抗原、抗血清最佳工作浓度分别为10ug/mL、1:12800,酶标二抗工作浓度为1:5000,最佳包被条件为4℃、24h包被;最佳封闭条件为37℃、1h; IFA兔高免血清稀释度为1:20,FITC标记羊抗兔IgG稀释度为1:10,感作时间为45min时,禽波氏杆菌特异性黄绿色荧光最清晰,非特异性荧光最弱。经临床检测证明该两种方法均具有简便、快速、特异性强的特点,为禽波氏杆菌病的流行病学、血清学调查和临床快速诊断奠定了基础。
试验二、禽波氏杆菌致病性相关毒素的提取及生物学特性检测
本试验对禽波氏杆菌内毒素进行了提纯,并首次粗提了禽波氏杆菌DNT及FHA。用制备的毒素对青紫兰健康家兔、Balb/c小鼠、雏鸡、SPF鸡胚、豚鼠进行动物试验,对禽波氏杆菌的致病性及本身的生物学特性进行研究探讨。同时建立起鸡胚气管环模型,验证气管细胞毒素的存在及其对气管纤毛上皮的致病作用。结果表明:禽波氏杆菌内毒素可使动物呈急性败血症病变。DNT能够致死小白鼠和雏鸡,并对肌肉组织产生损害。FHA只凝集豚鼠红细胞,对家兔和鸡的红细胞不产生凝集作用。禽波氏杆菌P5、P8株分别作10-8.5、10-8.3倍稀释,可使SPF鸡胚气管环接种0.1mL后50%出现气管环纤毛脱落,气管环粘膜出现凹陷、空洞,初步验证了气管细胞毒素的存在及禽波氏杆菌对气管纤毛的损伤机理。对禽波氏杆菌相关毒素的生物学特性的初探为研究禽波氏杆菌的保护性抗原提供了重要依据。
Bordetalla avium disease was a high-contacted infectious disease.In our country,it was discovered by Rui-liang Zhu in 1990.It could arouse that chicken-embryo death;low incubation;chicking acuting death and chicken opHthalmia so on.The infection rate of this disease was 10%～50%,it could bring up illness-chicken grew up lowly, the low redound upon of feedingstuff,and great economy loss.
Recent years, the immunity of outer-membrane-protein of bacteria in cell-wall was paid attention to by science workers.the test testified that OMP with well immunity could stimulate not only humoral immunity and cell immunity. The study indicates that the labelled antibody technique used Bordetella avium OMP is rapid, reliable and sensitive and it has the potential for use as an fast diagnosis for Bordetella avium.
The pathogenesis of Bordetella avium is related to its virulence factors. Presumably, these factors may play an important role in pathology and immunity. In order to solve problems mentioned above, Pure endotoxin、raw dermonecrotic toxin and fibrillae hemagglitinins of Bordetella avium were prepared and chicken embryo tracheal ring model was established for detecting biological characteristics of Bordetella avium.It preliminarity revealed relationship between Bordetella avium toxins and its pathogenicity.
The research divides two parts:
Part One: Preparation of High Titer Immuno Serum and Establishment of detection methods by Bordetella avium OMP with labelled antibody technique
In this study, out membrane proteins of B.avium strains P5 and P8 were prepared by improved Wooldridge method. We could determine contents of Bordetella avium OMP by Bradford, do SDS-PAGE electropHoresis of Bordetella avium OMP and prepare OMP immunity to immunity rabbits for preparation of high titer immuno serum. And then detections of indirect ELISA and indirect immunofluorescence were established by optimization and screening of conditions. The result showed that: the contents of Bordetella avium OMP was 320 ug/mL. The protein molecular weight of P5 and P8 strains is respectively 58、47、41、36、24KD, 58、47、41、38、21、16KD. The detection technique of labelled antibody was simple, rapid and strong specificity. It can be used to study on the epidemiology and serology of bordetella avium. This method laid a foundation of fast diagnosis for bordetella avium.
Part Two: Extraction and detection of biological characteristics of Bordetella avium related toxins
Pure endotoxin、raw dermonecrotic toxin and fibrillae hemagglitinins of Bordetella avium were prepared to used for animal test of rabbits、Balb/c mice、chicks、SPF chicken embryos、guinea pigs. Pathogenicity and biological characteristics of Bordetella avium were researched and explored by animal test. At the same time, chicken embryo tracheal ring model was established to verify the existence of tracheal cytotoxin and its pathogenic effects on tracheal cilia epithelium. The results showed that: endotoxin of Bordetella avium can effectively lead to acute septicemia lesions of animals. DNT caused little white rats、chicks death and muscle tissue damaged. FHA only agglutinated erythrocytes of guinea pigs, it had no agglutination to erythrocytes of rabbit and chicken. When Bordetella avium P5、P8 were diluted 10-8.5、10-8.3 times , abscission and injury of 50% of chicken embryo tracheal rings cilia could be caused by inoculating 0.1mL Bordetella avium.This result preliminarily verified the existence of tracheal cytotoxin and revealed pathogenic mechanism of Bordetella avium on tracheal cilia.The biological characteristics of these toxins played an important role for studing Bordetella avium vaccines.
引文
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