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1 VPp1,,, (, ) 摘要 : 为探究副溶血弧菌 (Vibrio parahaemolyticus) 的生物防治方法, 从水产品市场处污水中分离出一株副溶血弧菌噬菌体 VPp1 并借助噬菌斑形态电镜酶切等技术对其进行了分类鉴定, 同时测定了其裂解谱最佳感染复数一步生长曲线以研究其裂解性能分类鉴定结果表明, 其核酸是线型双链 DNA, 大小在 15 kb 左右具有一个正二十面体的头部, 头部直径大约为 44 nm, 无尾, 属盖噬菌体科 (Tectivirus); 裂解性能研究结果表明, 其在双层平板上培养 12 h 可形成中心清亮的噬菌斑, 周围有大而明显的晕环最佳感染复数为 , 潜伏期为 10 min, 裂解量为 90.3, 是符合条件的潜伏期短裂解量大的理想噬菌体, 可用作进一步的应用关键词 : 噬菌体 ; 副溶血弧菌 (Vibrio parahaemolyticus); 鉴定 ; 裂解性能中图分类号 : Q 文献标识码 : A 文章编号 : (2013) (Vibriosis),,,, (Vibrio parahaemolyticus) (Vibrio anguillarum) (Vibrio alginolyticus) (Vibrio harveyi) (Vibrio vulnificus) (Vibrio fluvialis) [1],, [2] [3],, [1],,,,,,,, (Bacteriophage) 96 / 2013 / 37 / 1, [4],,, 12 h,, 1 材料与方法 1.1 材料 Vibrio parahaemolyticus ( VP 17802), Fujino [5] 1953 (American type culture collection, ATCC) V. parahaemolyticus : ; : : ( ); (nycytx-50); (ZR2011CQ024) : (1987-),,,,, : , pengyongpy520@163.com;,, : , snow@ouc.edu.cn

平板上培养 12 h 可形成中心清亮的噬菌斑, 周围有大而明显的晕环最佳感染复数为 0.0001, 潜伏期为 10 min, 裂解量为 90.

2 VP VIB304 VP VIB461 VP VIB800 ; VP 17802(sj) F3-3 ; qdfsvp001, 16SrDNA E, SM 5.8 g NaCl, 2 g MgSO 4 7H 2 O, 50 ml 1 mol/l ph 7.5 Tris-HCl, ml, 1.2 方法 , 30 ml 50 ml r/min, 10 min, 5 ml 5 ml E, 100 µl VP 17802(OD 600 = 0.837), 37 C, r/min, 10 min, 0.22 µm,, 4 C, SM, 100 µl VP 17802(OD 600 = 0.837) 100 µl 5 ml 2216E,, 2216E,, [6], : 1 ml SM,, 4 C, SM, 100 µl 100 µl VP 17802(OD 600 = 0.837), 37 C 30 min, 5 ml,, 37 C SM,, 1.2.4,, 10 ml SM, 4 C, r/min, 15 min, 0.22 µm,, 4 C pfu/ml 1 ml r/min 59 min ( ),,, UNIP-10 ( ) , DNaseI RNaseA Mung Bean Nuclease, [7-8], : 20 μl pfu/ml,, 2~3 min,, 2%, 10 min,, 1.2.8, 100 µl 100 µl (OD 600 = 0.837), min, 5 ml,, 37, 30~300 VPp1 VP 17802(sj) F3-3 VP VIB304 VP VIB461 VP VIB800 qdfsvp (MOI ) , 100 µl 100 µl VP ( CFU/mL ) 10 ml 2216E, 37 C 10 h, 4000 r/min 15 min,, 0.22 µm,, Marine Sciences / Vol. 37, No. 1 /

837), 37 C 30 min, 5 ml,, 37 C 5 1.2.3 SM,, 1.2.4,, 10 ml SM, 4 C, 8 000 r/min, 15 min, 0.22 µm,, 4 C 1.2.5 10 9 pfu/ml 1 ml 12 000 r/min 59 min ( ),,, UNIP-10 ( ) 20 1.2.6, DNaseI RNaseA Mung Bean Nuclease, 1.

3 [9] 1 ml 1 ml VP (OD 600 = 0.837) 10 8 ml 2216E, 37 C 5 min, r/min 30 s,, 2216E 37 C 2216E, 37 C,, 30 min 5 min, 10 min,,, 2 结果 2.1 噬菌体的分离纯化及噬菌斑的形态观察 VPp1, 37 C 12 h, VP 17802, ( 1),,, pfu/ml 2 Fig. 2 The nucleic acid of phage VPp1 M1.1kb DNA ; A. VPp1 ; M2. λ-hind III digest DNA M1.1kb DNA ladder Marker; A. Phages nucleic acid; M2. λ-hind III digest DNA Marker 1 Fig. 1 The bacteriophage plaque of phage VPp1 2.2 噬菌体核酸的提取 ( 2),,, Quantity One VPp bp 2.3 噬菌体核酸类型的鉴定 ( 3), DNaseI, RNaseA, DNA, RNA, 3 Fig. 3 Determination of the nucleic acid type of bacteriophage M. 1kb DNA ; A. ; B. DNaseI ; C. RNaseA ; D. Mung Bean Nuclease M.1kb DNA ladder Marker; A. Phages nucleic acid; B. Phages nucleic acid mixed with DNaseI; C. Phages nucleic acid mixed with RNaseA; D. Phages nucleic acid mixed with Mung Bean Nuclease 98 / 2013 / 37 / 1

1kb DNA ladder Marker; A. Phages nucleic acid; M2. λ-hind III digest DNA Marker 1 Fig. 1 The bacteriophage plaque of phage VPp1 2.2 噬菌体核酸的提取 ( 2),,, Quantity One VPp1 15738.77 bp 2.

4 Mung Bean Nuclease, 3, DNA 2.4 噬菌体的电镜观察 ( 4), VPp1,, 44 nm ICTV [10], (Tectivirus) 4 VPp1 ( ) Fig. 4 Electron micrograph of phage VPp1 2.5 噬菌体裂解谱的测定 h VPp1 VP 17802, VP 17802(sj) VP VIB304, VP VIB304 VP VIB461 VP VIB800 VP 17802(sj) F3-3 qdfsvp 噬菌体最佳感染复数 (MOI 值 ) 的测定 1 1, ,,,, , 2.62± , VPp 噬菌体一步生长曲线的测定 VPp1 5 5, VPp1 10 min,, 10 min; 10~30 min,, VPp1 20 min; 表 1 噬菌体 VPp1 最佳感染复数的测定 Tab. 1 Determination of bacteriophage VPp1 MOI ( ) ( ) (pfu/ml) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± min,, : = / = / = 讨论 5 VPp1 Fig. 5 One-step growth curve of phage VPp1 4 5, 6,,,, Marine Sciences / Vol. 37, No. 1 /

03 10 10, VPp1 0.0001 2.7 噬菌体一步生长曲线的测定 VPp1 5 5, VPp1 10 min,, 10 min; 10~30 min,, VPp1 20 min; 表 1 噬菌体 VPp1 最佳感染复数的测定 Tab.

5 [11], [12],,,,, 2005 (ICTV) 5, [13] DNA,, I,,,,, [14],,,,,, [15],,,, VPp , [16] 0.1~10, Onanong [17] 0.01,, VPp1 3 : VPp1, 90.3,, : [1],,,. [J]., 2009, 28(8): [2],. [J]., 2001, 8(1): [3] Yicheng S, Chengchu L. Vibrio parahaemolyticus: A concern of seafood safety[j]. Food Microbiology, 2007, 24: [4],,,. [J]., 2004, 23(2): [5] Fujino T, Miwatani T, Yasuda J. Taxonomic studies on the bacterial strains isolated from cases of shirasu food-poisoning (Pasteurella parahaemolytica) and related microorganisms[j]. Biken Journal, 1965, 8(2): [6] 効, 穉. [M]. :, 1991: 10. [7],,. VP 10 [J]., 2003, 22(2): [8],, -II (Vibrio fluvialis-ii) VP 8 [J]., 2000, 20(1): [9]. O157 [D]. :, [10],. ICTV [J]., 2006, 21(1): [11],,. [J]., 2010, 29(5): [12]. SWBC-a [J]., 2009, 28(6): [13],,. [J]., 2007, 27(4): [14] Graschopf A, Blasi U. Functional assembly of the lambda S2 holin requires periplasmatic localization of its N2 terminus[j]. Arch Microbiol, 1999, 172: 31. [15]. K88 [D]. :, [16],,. [J]., 2007, 34(4): [17] Onanong P, Nuttaporn P, Nuttika S, et al. Isolation and characterization of a novel Podoviridae-phage infecting Weissella cibaria N22 from Nham, a Thai fermented pork sausage[j]. Food Microbiology, 2011(28): / 2013 / 37 / 1

Taxonomic studies on the bacterial strains isolated from cases of shirasu food-poisoning (Pasteurella parahaemolytica) and related microorganisms[j]. Biken Journal, 1965, 8(2): 63-71. [6] 効, 穉. [M].

6 Isolation, identification and lysis properties analysis of a Vibrio parahaemolyticus phage VPp1 PENG Yong, DING Yun-juan, LIN Hong, WANG Jing-xue (Food Safety Laboratory, College of Food Science and Engineering, Ocean University of China, Qingdao, , China) Received: Dec.,2, 2011 Key words: phage; Vibrio parahaemolyticus; identification; lysis properties Abstract: In order to develop an effective bio-controlling method against Vibrio parahaemolyticus, a virulent phage VPp1 was isolated from aquatic sewage water and identified by plaque shapes, restriction, electronic microscope and lysis properties analyses. The results revealed that VPp1 contains a linear double stranded DNA with 15kb in size and it has an icosahedral head with 44 nm in diameter and does not have a tail, suggesting that VPp1 belongs to the Tectivirus family. VPp1 forms plaques with transparent center and large halo after culture in double-layer agar plate for 12 hours and its optimum MOI value was A one-step growth experiment showed that the latent period and burst size were estimated at 10 min and 90.3 phage particles/infected cell, respectively. VPp1 could be widely used because of its short latent period and large burst size. ( 本文编辑 : 谭雪静 ) Marine Sciences / Vol. 37, No. 1 /

,2, 2011 Key words: phage; Vibrio parahaemolyticus; identification; lysis properties Abstract: In order to develop an effective bio-controlling method against Vibrio parahaemolyticus, a virulent

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