对虾WSSV病毒VP281基因的siRNA筛选研究

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...

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Published in西南农业学报 Vol. 24; no. 5; pp. 1992 - 1996
Main Author 黎铭 陈晓汉 陈秀荔 彭金霞 马春霞 蒋伟明 彭敏 赵永贞 杨春玲 李咏梅
Format Journal Article
LanguageChinese
Published 广西水产研究所遗传育种与健康养殖重点实验室,广西南宁,530021%广西兽医研究所,广西南宁,530001 2011
Subjects
RNA干扰
VP281
WSSV
对虾
VP281
RNA干扰
对虾
WSSV
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ISSN1001-4829
DOI10.3969/j.issn.1001-4829.2011.05.076

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Summary: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。
Bibliography:Shrimp; WSSV; VP281; RNA interference
51-1213/S
LI Ming,CHEN Xiao-han,CHEN Xiu-li,PENG Jin-xia,MA Chun-xia,JIANG Wei-ming,PENG Min,ZHAO Yong-zhen,YANG Chun-ling,LI Yong-mei（1.Key Laboratory of Aquatic Genetic Breeding and Healthy Aquaculture,Guangxi Institute of Fisheries,Guangxi Nanning 530021,China;2.Guangxi Veterinary Research Institute,Guangxi Nanning 530001,China）
Constructed a screen system for specific siRNA to VP281 and find the specific siRNA which would be useful in the research on RNAi to WSSV.According to the code sequence for VP281,a pair of primers was designed.The amplified VP281 was digested by restriction enzymes and the same as pEGFP-C1.An vitro vector named pEGFP-VP28 was constructed by connecting pEGFP-C1 and VP281.siRNA targeting VP281（VP281-siRNA1,VP281-siRNA2,VP281-siRNA3） were designed by software.Each siRNA was synthesized by chemical method and then were contransfected with pEGFP-VP281 into PK cell.The expression of pEGFP-VP281 was verified by Western blot,and the interference effect to
ISSN:1001-4829
DOI:10.3969/j.issn.1001-4829.2011.05.076