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取经人工注射感染了对虾白斑综合症病毒 40— 45h的凡纳对虾鳃组织 ,分离mRNA ,以mRNA为模板合成双链cDNA ,并克隆于PUC质粒的NotI/SalI位点 ,构建了 1 0 0 0余株对虾感染后期鳃细胞的重组cDNA克隆。重组质粒经PCR鉴定插入片段 ,DNA斑点杂交分析目的片段 ,测定了 2 0株对虾白斑综合症病毒的重组cDNA克隆的末端DNA序列 ,并对其进行了包含存在的开放阅读框架、启动区上游序列、编码产物的特性等分析。结果显示 :PCR产物在0 3— 1 6kb之间 ;大于 1kb的克隆中有 31 8%的克隆为白斑综合症病毒的重组cDNA克隆。已测序的不包含同源序列的 1 3株克隆中含有 1 4个开放阅读框 ,其中 1 1个上游可检出启动子基序 ,4个可检出启动子调制元件。ORF转译产物的特性基序分析显示 :有 2个ORFs可检出锌指基序 ,3个ORFs可检出亮氨酸拉链基序 ,2个ORFs可检出NTP结合基序 ,未检出核定位信号基序。
The gill tissues of P. vannamei infected with WSSV 40-45h were isolated by artificial injection. MRNA was synthesized and double-stranded cDNA was synthesized using mRNA as a template. The double-stranded cDNA was cloned into the NotI / SalI site of PUC plasmid to construct a 10 000 Recombinant cDNA Cloning of Gill Cells from the Late Strain of Penaeus. The recombinant plasmid was identified by PCR and inserted into the DNA fragment. The DNA fragment was analyzed by restriction endonuclease analysis, and the terminal DNA sequence of the 20 cDNA fragments of white spot syndrome virus (WSSVV) was determined. The open reading frame (ORF) , The nature of the encoded product and the like. The results showed that the PCR products were between 0 3 and 1 6 kb, and 31.8% of the clones larger than 1kb were the recombinant cDNA clones of WSSV. The 13 sequenced clones that did not contain homologous sequences contained 14 open reading frames, of which 11 could detect promoter motifs and 4 detectable promoter modulators. The analysis of the ORF translation product motif showed that two ORFs could detect zinc finger motifs, three ORFs could detect leucine zipper motifs, two ORFs could detect NTP binding motifs and no detectable nuclei Positioning signal motifs.