对虾白斑综合症病毒重组cDNA克隆的构建与分析

THE CONSTRACTION AND ANALYSIS FOR cDNA CLONES OF WHITE SPOT SYNDROME VIRUS OF SHRIMP

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。

Shrimp gills of Litopenaeus vannamei , which have been infected by White Spot Syndrome Virus of shrimp, were collected after injecting 40 45 hours. The Poly(A) +mRNA were selected from purified gill's total RNA using Oligo(dT) cellulose in a filter syringe format. The double strand cDNA were catalyzed by AMV reverse transcriptase and E. coli DNA polymerase | in combination with E. coli RNase H and E. coli DNA ligase using the mRNA as a template. After ligating of Size Fractionated cDNA to the Plasmid Vector PUC19 and introducing into E. coli , more than 1000 cDNA clones were harvest. We analysis the insert by PCR, analysis aim clones by dot blot hybridization and sequenced terminal DNA sequence of 20 cDNA of White Spot Syndrome Virus clones. We still analysis the Open Reading Frame of cDNA clones by some computer software and Web server, analysis upstream DNA sequence of ORF and the character of putative protein. We have look up the alignment DNA and protein of cDNA in NCBI database. The results of PCR shown that the PCR products are from 0.3kb to 1.6kb. There are 313 clones its products are more than 1kb, this occupied 31.5% of all cDNA clones. There are 31.8% clones are WSSV cDNA clones by DNA dot blot hybridization. 14 full or not full ORFs have been found in 13 clones. The upstream DNA sequence analysis shown that there are 7 ORFs have early promoter motifs and there are 4 ORFs have later promoter motifs, among of them there are 3 ORFs which have early and later promoter motifs too. And the enhancer motifs can be found in 4 ORFs, the G+C rich sequence is the common motif. The analysis of the special protein motif of ORF shown that the Zinc finger motif can be found in 2 ORFs, Leucine Zipper motif can be found in 3 ORFs, the NTP binding motif can be found in 2 ORFs, and the nuclear localization signal motif can't be found in 14 ORFs. ORF S13 has alignment protein by BLAST in NCBI database.