摘要
利用RNA干扰在线生物学软件分析猪瘟病毒NS3基因,设计针对NS3基因不同位置的小发卡RNA(short hairpin RNA,shRNA)干扰序列(其结构特征为正链(19nt)-环(9nt)-负链(19nt))。化学合成这些序列,并退火连接为双链干扰片段,将双链干扰片段定向克隆到干扰载体中,构建重组载体pGene-NS3-1,pGene-NS3-2和pGene-NS3-3,转化DH5α大肠杆菌感受态细胞,对转化产物进行kan+筛选,碱裂解法提取重组质粒,将酶切鉴定为阳性的质粒测序。结果显示,双链干扰片段克隆方向正确,序列中未出现核苷酸的插入,缺失等突变现象。表明靶向猪瘟病毒NS3基因shRNA干扰载体构建成功。
19nt interferencing sequences for selecting different positions of NS3 gene were designed for Sence-Loop-Antisence by using biology software after analysising NS3 gene of classic swine fever virus. Then ,these sequences of chemical synthesis were annealed for double strands interference segment ,which was made directional cloning to interferecing plasmid to construct recombinat plasmids pGene-NS3-1, pGene-NS3-2 and p-Gene-NS3. The results indicated that segments were cloned rightly and there wasn＇t nucleotide insertion and nucleotide deletion, and recombinant shRNA interference plasmids targeting to NS3 gene of CSFV was successfully constructed,which was basic for researching NS gene.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2007年第3期7-10,共4页
Journal of Northwest A&F University(Natural Science Edition)
基金
国家自然科学基金项目(30270988)
西北农林科技大学研究生教育创新计划项目(05Ych019)
