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构建一个对虾WSSV VP281基因的siRNA筛选体系,获取有效siRNA,为进一步开展siRNA抗WSSV研究建立基础。根据已报道的VP281基因编码序列,设计并合成一对引物,扩增出带双酶切位点的VP281。对VP281和质粒pEGFP-C1分别酶切后进行连接,获取表达载体pEGFP-VP281。利用专业软件设计3对靶向VP281的siRNA(VP281-siRNA1﹑VP281-siRNA2﹑VP281-siRNA3),并合成siRNA,将3种siRNA分别与pEGFP-VP281共转染PK细胞。采用Western blot方法检测GFP-VP281融合蛋白的表达,半定量RT-PCR方法检验siRNA抑制VP281基因转录的效果。结果显示,pEGFP-VP281可在BHK细胞正常表达融合蛋白GFP-VP281。3对siRNA对GFP-VP281的mRNA转录均有不同程度的干扰效果,siRNA2的干扰效果最为显著。构建针对WSSV-VP281基因的siRNA筛选体系,初步验证了该系统的有效性,为开展siRNA抗WSSV研究建立了基础。
To construct a siRNA screening system for shrimp WSSV VP281 gene and obtain effective siRNAs, and to lay the foundation for further research on anti-WSSV siRNA. According to the reported VP281 gene coding sequence, a pair of primers was designed and synthesized to amplify VP281 with double restriction enzyme sites. VP281 and plasmid pEGFP-C1 were digested and ligated respectively, and the expression vector pEGFP-VP281 was obtained. Three pairs of siRNA targeting VP281 (VP281-siRNA1, VP281-siRNA2 and VP281-siRNA3) were designed and synthesized by professional software. The three siRNAs were cotransfected into PK cells with pEGFP-VP281 respectively. The expression of GFP-VP281 fusion protein was detected by Western blot and the effect of siRNA on VP281 gene transcription was tested by semi-quantitative RT-PCR. The results showed that pEGFP-VP281 could interfere with mRNA expression of GFP-VP281 in BHK cells with different levels of GFP-VP281.3, and siRNA2 had the best interference effect. The construction of a siRNA screening system targeting WSSV-VP281 gene initially validated the system and laid the foundation for the development of an siRNA against WSSV.