【摘要】 急性病毒性坏死症病毒(Acute Virus Necrobiotic Virus,AVNV)已被证实是一种对养殖栉孔扇贝(Chlamys farreri)危害极大的致病病原。本研究应用基因克隆技术对AVNV基因组进行cDNA合成、克隆,并对部分克隆进行序列分析。采用差速和蔗糖密度梯度离心法,分离纯化AVNV。用Trizol试剂抽提病毒RNA,采用随机引物和Oligo(dT)双引物法,SuperScriptⅡ反转录酶合成cDNA,并克隆于pUC118质粒上,利用蓝白斑筛选阳性克隆子,PCR法快速鉴定重组质粒。经筛选得到2个阳性克隆23#、52#,插入片段大小约为1 200 bp和800 bp。测序结果与GenBank的比对分析显示,尚未有类似的序列报道,提示该病毒可能为一种新发现的病毒。更多还原

【Abstract】 The scallop,Chlamys farreri,is one of the major species cultured in North China,and its culture in commercial scale has been performed more than 20 years.The highest production(about 10~6 t) has been achieved in 1997.However,the great expansion and intensification have induced the occurrence of disease since 1990’s,especially the disease has been becoming epizootics since 1997 in Shandong and Liaoning provinces.It is believed that the disease so called Acute Viral Necrotic Disease’(AVND) has been becoming the major limiting factor in the development of the scallop industry,and has stroke the economic process in the coastal region.In order to understand molecular biologic characteristics of Acute Viral Necrotic Virus(AVNV) and establish molecular diagnostic methods, cloning and sequenc analysis had been undertaken.AVNV was isolated and purified from moribund scallop through differential centrifugation and discontinuous sucrose gradient centrifugation.The total RNA was extracted from AVNV with Trizol reagent.The cDNA was synthesized with random hexamers and Oligo(dT) method.After ligating of Size-Fractionated cDNA to the plasmid vector pUC118 and introducing into E.coli DH5α,the recombinant plasmids were screened by blue and white colonies and checked by PCR amplification.Two positive clones,23~# and 52~#,were obtained,size of insert segment were about 1?200?bp and 800?bp respectively.The partial cDNA clones were sequenced and analyzed.According to the results of nucleotide sequence analysis,none of 23~# or 52~# clone shows significant homology with those of other known sequences in GenBank.The results indicate AVNV would be a new virus.更多还原