甲型副伤寒沙门菌噬菌体的分离及其生物学特性的分析  

Isolation and biological characters of bacteriophages of Salmonella paratyphi A

作　　者：毛普加 冯金[1] 洪愉[2] 黄芬[1] 井申荣[1] 曾韦锟[1] 

机构地区：[1]昆明理工大学医学院,云南昆明650500 [2]成都军区昆明总医院,云南昆明650532

出　　处：《中国生物制品学杂志》2014年第4期458-462,466,共6页Chinese Journal of Biologicals

基　　金：国家自然科学基金项目(31160193);云南省教育厅科学研究基金项目(2010Y398);云南省应用基础研究面上项目(2010ZC055;2012FB135)

摘　　要：目的分离甲型副伤寒沙门菌（副甲菌）噬菌体（副甲噬菌体），并分析其生物学特性。方法从昆明某医院污水中分离副甲噬菌体，并采用噬斑形成法检测其滴度；透射电镜下观察浓缩纯化后的副甲噬菌体形态；分析副甲噬菌体对不同pH（1～12）和不同温度（40、50、60、70、80℃）作用的敏感性；培养副甲菌和副甲噬菌体，以不同培养时间为横坐标，对应的噬菌体滴度为纵坐标，绘制一步生长曲线，并测定爆发量、最佳MOI及副甲菌突变率；提取副甲噬菌体基因组，酶切分析副甲噬菌体核酸，SDS-PAGE分析副甲噬菌体外壳蛋白；采用热酚法提取脂多糖（1ipopolysac-chafide，LPS），采用苯甲基磺酰氟（phenylmethanesulfnnvl fluoride，PMSF）法提取外膜蛋白（outer membrane protein，OMP），初步分析副甲噬菌体的受体。结果在医院的污水中分离出的副甲噬菌体，经双层平板计数可见大小不一、透明的噬斑，滴度在109.10mPFU之间，透射电镜观察可见长尾和一个头部；副甲噬菌体在pH值3～10之间滴度较高，40℃以上，随温度上升滴度明显降低，在80℃几乎无活性副甲噬菌体存在；副甲噬菌体感染潜伏期为20min，爆发期为50min，爆发量129，最佳MOI为0.2，副甲菌突变率为5.8×10^-7～2.2×10^-6；副甲噬菌体基因组的单酶切产物均可见33bp的特异条带，SDS-PAGE分析可见4条主带；LPS、OMP能与副甲噬菌体结合，是副甲噬菌体的受体。结论副甲噬菌体为长尾噬菌体，基因组为dsDNA，大小为33bp，最佳MOI为0.2，受体位点为LPS和OMP。Objective To isolate the bacteriophages of Salmonella paratyphi A and analyze its biological characters. Methods The bacteriophages of X paratyphi A was isolated from sewage, determined for titer by plaque formation assay, observed for morphology by electron microscopy after concentration and purification, and analyzed for sensitivities to pH value （ 1 ～ 12） and temperature （40, 50, 60, 70 and 80℃）. S. paratyphi A and its bacteriophages were cultured, of which the onestep growth curves were plotted using culture time as abscissa and the corresponding bacteriophage as ordinate, and the outbreak level of bacteriophages, optimal MOI and mutation rate of S. paratyphi A were determined. The genome of bacteriophages was extracted and analyzed for DNA by enzyme digestion. The capsid protein of S. paratyphi A was analyzed by SDS-PAGE. Lipopolysaccharide （LPS） was extracted by hot phenol method, while the outer membrane protein （OMP） by phenylmethanesulfonyl fluoride （PMS） method, and the receptor of S. paratyphi A was analyzed preliminarily. Results Paratyphi A bacteriophage was isolated from sewage of the hospital, of which a long tail and a head were observed by electron microscopy, and transparent plaques at various sizes on double layer counting plate, at titers of 10^9 ～ 10^10 PFU. The titer of bacteriophages was high at pH 3 - 10, while decreased significantly at temperatures of more than 40℃ with the increasing temperature. However, at 80℃, few active bacteriophages were observed. The incubation period, outbreak period, outbreak level and optimal MOI of bacteriophages were 20 min, 50 min, 129 and 0.2 respectively, while the mutation rate of S. paratyphi A was 5.8 × 10^-7 ～ 2. 2 × 10^-6. The specific band at a length of 33 bp was obtained by digestion of S. paratyphi A with EcoR I and Hind m , and four major bands were observed on SDS-PAGE profile. Both LPS and OMP were the receptors of S. paratyphi A. Conclusion Paratyphi A bacteriophages were long tail phage, of which the genome

关 键 词：沙门菌 甲型副伤寒 细菌噬菌体 生物学特性 

分 类 号：R378.23[医药卫生—病原生物学] Q939.48[医药卫生—基础医学]