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为了寻找对虾抗桃拉病毒(TSV)相关基因信息,研究对虾抗TSV分子机理。应用抑制性差减杂交(SSH)技术,构建TSV感染后存活凡纳宾对虾与未感染凡纳宾对虾的基因差异表达文库,并通过相对定量PCR以及斑点杂交分别对SSH的效率以及文库的可靠性进行验证。结果表明,SPR凡纳宾对虾和SPF凡纳宾对虾攻毒后的死亡率分别为12%和90%;在SSH系统中,相对于未进行杂交的cDNA样品,杂交后的cDNA样品的β-肌动蛋白(β-actin)几乎没有被检测出,说明SSH操作系统是高效率的。通过文库构建则获得正负2个文库,其中正库含阳性克隆1051个,负库含阳性克隆580个;而经斑点杂交验证,共获差异表达克隆321个,其中攻毒组高表达克隆270个,未攻毒组高表达克隆51个。可见,SPR凡纳宾对虾具有较强的抗TSV能力,以之为基础构建的对虾基因差异表达文库具有高度的可靠性,由文库得到的基因信息对获取对虾抗(TSV)相关基因具有潜在的应用价值。
In order to find out the gene information of shrimp against Taura virus (TSV), the shrimp anti-TSV molecular mechanism was studied. Suppression subtractive hybridization (SSH) technique was used to construct a gene differential expression library that survived TSV infection and which did not infect E. vannamei. The efficiency of SSH and the reliability of the library were determined by relative quantitative PCR and dot blot hybridization authenticating. The results showed that the death rates of P. vannamei and P. vannamei were 12% and 90%, respectively. In the SSH system, compared with the non-hybridized cDNA samples, the β- Actin was barely detectable, indicating that the SSH operating system is highly efficient. Positive and negative 2 libraries were obtained by library construction, including 1051 positive clones in the positive database and 580 positive clones in the negative library. 321 positive clones were identified by dot blot hybridization, in which the cloned high expression clones 270 There were 51 clones highly expressed in the untreated group. It can be seen that the SPR Vannamei shrimp possesses strong anti-TSV ability, and the shrimp gene differential expression library constructed on the basis of the SPR is highly reliable. The gene information obtained from the library has potential potential for obtaining the shrimp anti-TSV related gene Value.