CAJ | 학술논문

通过在E． coli菌液中加入土壤悬浮液共培养获得噬菌体，利用双层平板法反复纯化4次后得到单一清晰的噬菌斑。将单个噬菌斑加入对数早期的E． coli菌液中增殖培养后利用核酸抽提试剂盒提取总核酸，并分别用DNaseⅠ和RNaseA对总核酸进行消化，利用限制性内切酶HindⅢ和EcoRⅠ对其进行酶切；增殖后的噬菌体液进行噬菌体颗粒浓缩，取浓缩液负染后置于电子显微镜下确定噬菌体的形态特征，并进行SDS－PAGE电泳分析，确定其蛋白条带；同时测定其他生物学特性，包括滴度、最佳感染复数、一步生长曲线以及其对温度、pH、紫外线、氯仿的敏感性等。结果表明：分离并纯化出1株E． coli烈性噬菌体，命名为ΦTRI?1，形态特征：头部为二十面体的立体对称，直径约为60 nm，有一长尾，长约200 nm；其核酸属于双链DNA，按照国际病毒分类委员会分类标准，其属于有尾噬菌体目（ Caudovirales），长尾噬菌体科（ Siphoviridae）的烈性噬菌体。生物学特性测定结果显示ΦTRI?1对E． coli的最佳感染复数为0?1，潜伏期约为20 min，爆发期约为60 min，裂解量约为38；ΦTRI?1在40℃时活性最高，在20～70℃活性均较强，80℃时活性丧失；其酸碱耐受力较强，在偏碱环境下活性相对更高；对紫外线有一定的耐受能力；对氯仿敏感，浓度5％的氯仿即可致其失活。分离到的噬菌体属于有尾噬菌体目，长尾噬菌体科，综合其各项生物学特性可知其潜伏期短，裂解能力强，环境适应能力强，具有很好的杀菌效果，具有开发为抗E． coli菌剂的潜力。
통과재E． coli균액중가입토양현부액공배양획득서균체，이용쌍층평판법반복순화4차후득도단일청석적서균반。장단개서균반가입대수조기적E． coli균액중증식배양후이용핵산추제시제합제취총핵산，병분별용DNaseⅠ화RNaseA대총핵산진행소화，이용한제성내절매HindⅢ화EcoRⅠ대기진행매절；증식후적서균체액진행서균체과립농축，취농축액부염후치우전자현미경하학정서균체적형태특정，병진행SDS－PAGE전영분석，학정기단백조대；동시측정기타생물학특성，포괄적도、최가감염복수、일보생장곡선이급기대온도、pH、자외선、록방적민감성등。결과표명：분리병순화출1주E． coli렬성서균체，명명위ΦTRI?1，형태특정：두부위이십면체적입체대칭，직경약위60 nm，유일장미，장약200 nm；기핵산속우쌍련DNA，안조국제병독분류위원회분류표준，기속우유미서균체목（ Caudovirales），장미서균체과（ Siphoviridae）적렬성서균체。생물학특성측정결과현시ΦTRI?1대E． coli적최가감염복수위0?1，잠복기약위20 min，폭발기약위60 min，렬해량약위38；ΦTRI?1재40℃시활성최고，재20～70℃활성균교강，80℃시활성상실；기산감내수력교강，재편감배경하활성상대경고；대자외선유일정적내수능력；대록방민감，농도5％적록방즉가치기실활。분리도적서균체속우유미서균체목，장미서균체과，종합기각항생물학특성가지기잠복기단，렬해능력강，배경괄응능력강，구유흔호적살균효과，구유개발위항E． coli균제적잠력。
Phages were screened by co?cultivating of E. coli bacteria liquid and suspension of soil, and a single clear plaque was obtained after 4?time purification using two layer plating method. The single plaque was added into logarithmic phase E. coli bacteria liquid to cultivate phage culture. The total nucleic acid of the phage was extracted using the Phage DNA extraction kit, and digested with DNaseI, RNaseA, HindⅢ and EcoRⅠ. At the same time, morphological properties of the phage were determined with electron microscope after phage particles concentrated. Coat proteins of phage particles were analyzed by SDS-PAGE. And other biological properties including titer, MOI, one? step growth curve, and sensitivity to temperature, pH, ultraviolet ray and chloroform were deter?mined. A strain of lytic E. coli phages namedΦTRI?1 was isolated and purified. The electron micro?scope results showed that phage particles had icosahedra head with 60 nm in diameter, and a long tail about 200 nm and nucleic acid digestion showed it was a double?stranded DNA. According to classification standards of international commission on virus classification, it belonged to the order of Cuadovirales, the family of Siphoviridae. Determination of biological properties showed that multi?plicity of infection ( MOI) of the Escherichia coli phage was 0?1 and its growth curve showed that incubation period was 20 min, time of burst phase was 60 min, and burst size was 38. Activity of E. coli phage was higher between 20 ℃ and 70 ℃ and reached the maximum when temperature was 40 ℃, but when temperature was 80 ℃ the phage was inactivated. And it had strong receivable a?bility of pH changes and relatively higher activity in the alkali environment. The E. coli phage was not particularly sensitive to ultraviolet ray, but sensitive to chloroform, and 5% concentration of chloroform could cause inactivation. The E. coli phage isolated in this experiment belonged to the or?der of Cuadovirales, the family of Siphoviridae, and it had a shorter incubation period, a stronger sterilization ability and a stronger adaption to the environment, so it has the potential to be developed as a new preparation to control E. coli bacterium disease.