| 摘要: |
| 于1994和1995两年的7月从青岛崂山上马镇养虾场中染病的中国对虾中分离纯化出一种C型杆状病毒。为建立中国对虾病毒的鉴别和检测技术，利用随机扩增多态性DNA技术对电泳纯化的病毒DNA进行分析和研究。将病虾匀浆、经差速离心和蔗糖密度梯度离心纯化获得完整病毒粒子；从病毒中提取DNA后再进行电泳纯化，收集长度完整、均一的病毒DNA；在其他参数保持不变的情况下，对Operon生产的10碱基随机引物K试剂盒进行扩增试验。结果显示：1．病毒DNA有两种存在形式；2．K试剂盒中有2个引物能将病毒DNA扩增出长度不同的产物，其中编号为OPK—01的随机引物可产生多态性DNA片段；3．1995年中国对虾杆状病毒的RAPD分析结果与1994年相同，证实1994和1995两年度引起中国对虾疾病的病原是同一种杆状病毒。由此可以认为：随机扩增多态性DNA技术能将中国对虾C型杆状病毒扩增出多态性DNA片段而达到病毒鉴别和诊断的目的。 |
| 关键词: 中国对虾 杆状病毒 随机扩增多态性DNA |
| DOI： |
| 分类号: |
| 基金项目:国家攀登计划“B”资助项目，PDB6－6－1号 |
附件 |
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| RANDOM AMPLIFIED POLYMORPHIC DNA OF A KIND OF C TYPE BACULOVIRUS PURIFIED FROM PENAEUS CHINENSIS |
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Kong Jie1, Shi Tuo1, Liu Ping1, Han Lingling2, Xu Huaishu2, Xiang Jianhai3
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1.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;2.College of Marine Life Sciences, Ocean University of Qingdao, Qingdao 266003;3.Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071
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| Abstract: |
| A kind of type C baculovirus was detected and purified from diseased shrimps, Penaeus chinensis collected from Laoshan shrimp farms, Qingdao in July 1994 and 1995. In order to establish the identification and detection technique, randomly amplified polymorphic DNA(RAPD) was used to analyze the virus DNA extracted from the virus itself.
Morphologically intact baculovirus and nucleocapsids were isolated and purified by different centrifugations of 6000 r/min to 10000 r/min and density gradient centrifugations on 10%-50% sugrose at 25 000 r/min from cepholothorax homogenate. Viral genomic DNA was extracted from purified virions with a TE buffer (10 mmol/L Tris-HCl, 100 mmol/L EDTA, pH = 8.0) containing 0.5% (W/V) SDS and 200 μg/ml proteinase K, at 55°C for l.5h, followed by phenol-chloroform extraction and ethanol precipitation. The DNA was electrophoresed in 0.7% low-melting point agrose gels (Sigma) to recover the main band. The DNA concentration was estimated by electrophoresis.
Ten base random primers of Operon kit K were tested individually under the same experimental conditions. Random amplification of viral DNA was performed in a 25 μl reaction mixture containing 50--100 ng template DNA, 10 mmol/L Tris-HCl, pH=8.3, 50 mmol/L KCl, 2 mmol/L MgCl2, 0.001% gelatin, 200 μmol/L of each deoxynucleotide triphosphate, 15 pmol of each primer, and 2 units of Tag DNA polymerase. The reactions were performed by 45 cycles of 94°C denaturation, 33°C annealing and 72°C polymerization, respectively, for 1 min. A complete extension was done by 72C for 10 min. An aliquot of 20 μl of the prepared samples was electrophoresed at 4 V/cm for 1.5 h in a 1.5% agarose gel, followed by staining with ethidium bromide. DNA fragment was visualized and photographed under ultraviolet light.
The results show that : 1) the viral DNA extracted from the baculovirus existed in 2 forms; 2) two random primers in Operon kit K could produce different products in length from viral DNA, and one of which, designated OPK-01 primer, could produce polymorphic and reproducible fingerprint; and 3) RAPD provided the same results for viral DNA. The front and back bands recovered from electrophoresing viral DNA respectively produced similar RAPD fingerprints corresponding to the two separate years. It is concluded that the baculoviruses purified from diseased P. chinensis in 1994 and 1995 are the same species. The results of this study indicate that RAPD is a potentially powerful technique for shrimp baculovirus identification and diagnosis because of its polymorphism and reproducibility. |
| Key words: Penaeus chinensis, Baculovirus, RAPD |
