致病性坏死梭杆菌FN5株白细胞毒素特性研究
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摘要
本研究首次利用切向流技术从国内分离的致病性牛源坏死梭杆菌(Fusobacterium necrophorum, FN) FN5株培养物上清液中分离获得天然白细胞毒素(leukotoxin,Lkt),并在体外对该毒素的生物化学和生物学特性及免疫保护特性进行研究。研究内容如下:
首先,运用PCR技术鉴定出FN5的基因型为Fnn亚型;其次,通过菌落平板计数测得FN5的对数生长期出现在接种后8~10 h并建立坏死梭杆菌Lkt活性体外测定方法,并在此基础上考察毒素贮藏温度、细菌培养时间等因素对上清液白细胞毒性的影响。第三,利用切向流技术浓缩上清液获得粗提Lkt,免疫印迹试验结果证实在粗提Lkt中存在抗重组Lkt BSBS兔血清的抗原性物质。通过动物实验证实粗提白细胞毒素具有很强的免疫原性,但粗提Lkt对实验动物同样也表现出很强的致病性。
结合国外关于白细胞毒素分子量的报道,利用超滤技术对粗提白细胞毒素进行精细纯化获得纯化白细胞毒素,并再次通过动物实验和透射电子显微镜对粗提白细胞毒素和纯化白细胞毒素的毒性和免疫原性差异进行比较。结果表明,纯化白细胞毒素毒性弱于粗提白细胞毒素,并且纯化白细胞毒素表现出很强的免疫原性。另外,纯化白细胞毒素可以诱导牛血多形核白细胞出现凋亡和坏死。该研究为白细胞毒素亚单位疫苗研制提供了基础数据。
This study successfully isolated and purified the natural leukotoxin from the culture supernatant of the high pathogenic bovine Fusobacterium necrophorum(FN) 5 strains by Cross-flow filtration, and the biochemical and biological characteristics and its immunoprotection of the leukotoxin were studied in vitro. Contents are as follows:
The subspecies of FN5 was identified as subsp. necrophorum by PCR, the logarithmic phase of FN5 was determined by using the colony counting.The leukotoxicity of the cell-free culture supernatant of FN5 on bovine polymorphonuclear neutrophils leukocytes (PMNs) was determined by WST dye-reduction assay. The effect of store temperature and growth phase of bacterium on the leukotoxicity of culture supernatant was also examined.
The crude leukotoxin was collected by cross-flow filtration apparatus ( Sartorious Stedium Biotech) at 4℃. SDS-PAGE indicated that the leukotoxin is highly unstable, for it degradated into several high-molecular-weigh polypeptides; and these proteins can recognized by the antiserum raised against recombinant FN4 leukotoxin truncated fragment BSBSE in western blot.The toxicity and the immunoprotection of crude leukotoxin were examined. It can cause ribbits to death, and also can protect host from the formation of liver abscesses even with experimental challenage with viable bacteria.
Crude leukotoxin was polished by ultrafiltration, which retain the proteins that molecule weight greater than 100 kDa. The difference of pathogencity and immunoprotection between the crude leukotoxin and the purified leukotoxin were tested by animal experiment and transmission electron microscope (TEM), recpectively. The result revealed that the pathogencity of the polished leukotoxin was lower than crude leukotoxin, but the immunoprotection was similar.
The pathological changes of the bovine PMNs treated with crude leukotoxin and polished leukotoxin, respectively, show the characteristics that consist with cells undergoing apoptosis, and the leukotoxicity of polished leukotoxin was lower than crude leukotoxin.
The results from this study are suggestive of the immunogenicity and protective effects in rabbit and mice of the culture supernatant of FN5 containing leukotoxin, and this data can provided the foundation of the theory to prepare for a new leukotoxin subunit vaccine to immunoprophylaxis the liver abscesses and foot rot formation of cattle
首先,运用PCR技术鉴定出FN5的基因型为Fnn亚型;其次,通过菌落平板计数测得FN5的对数生长期出现在接种后8~10 h并建立坏死梭杆菌Lkt活性体外测定方法,并在此基础上考察毒素贮藏温度、细菌培养时间等因素对上清液白细胞毒性的影响。第三,利用切向流技术浓缩上清液获得粗提Lkt,免疫印迹试验结果证实在粗提Lkt中存在抗重组Lkt BSBS兔血清的抗原性物质。通过动物实验证实粗提白细胞毒素具有很强的免疫原性,但粗提Lkt对实验动物同样也表现出很强的致病性。
结合国外关于白细胞毒素分子量的报道,利用超滤技术对粗提白细胞毒素进行精细纯化获得纯化白细胞毒素,并再次通过动物实验和透射电子显微镜对粗提白细胞毒素和纯化白细胞毒素的毒性和免疫原性差异进行比较。结果表明,纯化白细胞毒素毒性弱于粗提白细胞毒素,并且纯化白细胞毒素表现出很强的免疫原性。另外,纯化白细胞毒素可以诱导牛血多形核白细胞出现凋亡和坏死。该研究为白细胞毒素亚单位疫苗研制提供了基础数据。
This study successfully isolated and purified the natural leukotoxin from the culture supernatant of the high pathogenic bovine Fusobacterium necrophorum(FN) 5 strains by Cross-flow filtration, and the biochemical and biological characteristics and its immunoprotection of the leukotoxin were studied in vitro. Contents are as follows:
The subspecies of FN5 was identified as subsp. necrophorum by PCR, the logarithmic phase of FN5 was determined by using the colony counting.The leukotoxicity of the cell-free culture supernatant of FN5 on bovine polymorphonuclear neutrophils leukocytes (PMNs) was determined by WST dye-reduction assay. The effect of store temperature and growth phase of bacterium on the leukotoxicity of culture supernatant was also examined.
The crude leukotoxin was collected by cross-flow filtration apparatus ( Sartorious Stedium Biotech) at 4℃. SDS-PAGE indicated that the leukotoxin is highly unstable, for it degradated into several high-molecular-weigh polypeptides; and these proteins can recognized by the antiserum raised against recombinant FN4 leukotoxin truncated fragment BSBSE in western blot.The toxicity and the immunoprotection of crude leukotoxin were examined. It can cause ribbits to death, and also can protect host from the formation of liver abscesses even with experimental challenage with viable bacteria.
Crude leukotoxin was polished by ultrafiltration, which retain the proteins that molecule weight greater than 100 kDa. The difference of pathogencity and immunoprotection between the crude leukotoxin and the purified leukotoxin were tested by animal experiment and transmission electron microscope (TEM), recpectively. The result revealed that the pathogencity of the polished leukotoxin was lower than crude leukotoxin, but the immunoprotection was similar.
The pathological changes of the bovine PMNs treated with crude leukotoxin and polished leukotoxin, respectively, show the characteristics that consist with cells undergoing apoptosis, and the leukotoxicity of polished leukotoxin was lower than crude leukotoxin.
The results from this study are suggestive of the immunogenicity and protective effects in rabbit and mice of the culture supernatant of FN5 containing leukotoxin, and this data can provided the foundation of the theory to prepare for a new leukotoxin subunit vaccine to immunoprophylaxis the liver abscesses and foot rot formation of cattle
引文
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[2]陆承平.兽医微生物学[M].中国农业出版社,第三版, 2001, 297-298.
[3]陈立志,王克坚,杨福合,等.坏死梭杆菌专用高效培养基及其制备方法:中国,200910066772.4
[4] C. Fievez. Comparative study of strains of sphaerophorus isolated from manand animals [J]. European Academic Press. Brussels. 1963, 20: 170-183.
[5] Nicholson, L. A.,Morrow, C.J., Corner, L.A. et al., Phylogenic relationship of Fusobacterium necrophorum A, AB, and B biotypes based upon 16s rRNA gene sequence analysis. International Journal of Systematic Bacteriology, 1994,44, 315-319.
[6] Shinjo T, Hiraiwa K, Miyazato S.,Recognition of biovar C of Fusobacterium necrophorum (Flugge)Moore and Holdeman as Fusobacterium pseudonecrophorum sp. nov.,nom.rev.(ex Pre′vot 1940). Int J Syst Bacteriol, 1990, 40: 71～73
[7] Shinjo, T., Shirakihara, M., Iwata, T., Hemagglutinin activity of Fusobacterium necrophorum with erythrocytes of various animals. Bull. Facul. Agricul. 1980, 27, 403-407.
[8] Nagai, S., Kanoe, M., Toda, M., Purification and partial characterization of Fusobacterium necrophorum hemagglutinin. Zentralbl. Bakteriol. Mikrobiol. Hyg. A. 1984, 258, 232-241.
[9] Kanoe, M., Koyanagi, Y., Kondo, C., et al., Location of hemagglutinin in bacterial cells of Fusobacterium necrophorum subsp. necrophorum. Microbios1998, 96, 33-38.
[10] Miyazato, S., Shinjo, T., Yago, H., Nakamura, N., Fimbriae (Pili) detected in Fusobacterium necrophorum. Jpn. J. Vet. Sci. 1978, 40, 619-621.
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