转录靶向猪瘟病毒3′-UTRshRNA细胞株干扰猪瘟病毒增殖的检测  

Detection of the Interference to the Reproduction of CSFV by PK-15 Cell Strains Transcribing shRNA Targeted to the 3′-UTR

作　　者：郭抗抗[1] 徐浩[1] 张彦明[1] 张为民[1] 冶贵生[1] 熊奎州[1] 谢林红[1] 党如意[1] 

机构地区：[1]西北农林科技大学动物医学院,杨凌712100

出　　处：《畜牧兽医学报》2008年第6期746-751,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金项目(30270988)

摘　　要：对筛选的转录猪瘟病毒石门株3-′UTR shRNA的PK-15细胞株P-1（114-132位）、P-2（136-154位）和P-3（209-227位）分别接种猪瘟病毒（CSFV）,在接毒后第72和96小时用半定量反转录-聚合酶链式反应（RT-PCR）检测CSFVNS3基因及-βactin基因,以研究构建细胞株在转录水平上对CSFV增殖干扰的效果;接毒后第72、96和148小时以酶联免疫吸附试验（ELISA）方法检测细胞NS3蛋白表达量,通过OD490值分析构建细胞株在病毒蛋白表达水平上对CSFV增殖的影响。检测结果表明：（1）接毒后第72和96小时P-1、P-2和P-3细胞株中NS3基因的量明显减少,与接毒的空白PK-15细胞比较差异显著（P〈0.05）,说明P-1、P-2和P-3细胞株转录的shRNA能干扰CSFV RNA的转录;（2）接毒后第72和96小时P-1、P-2、P-3细胞株中NS3蛋白的表达量明显少于接毒的PK-15细胞（P〈0.05）,说明转录的shRNA在病毒蛋白水平上能干扰CSFVNS3基因的表达,但接毒后第148小时干扰效果有所降低。TheP-1（114-132 sites）, P-2（136-154 sites） and P-3（209-227 sites） PK-15 cell strains transcribing shRNA targeted to the classical swine fever virus（CSFV） 3＇-UTR were infected with CSFV shimen strain respectively. The NS3 and β-actin gene of infected cells were detected by semi-quantitive reverse transcription polymerase chain reaction（RT-PCR） at the 72nd and 96th hour post-infection, to study the influence of transcribing shRNA on the reproduction of CSFV at RNA level. At the same time , the NS3 protein of cells was detected by enzyme linked immunosorbent assay（ELISA） at the 72nd, 96th and 148th hour post-infection to estimate the influence at protein level by comparing the optical density at 490 nm. Results showed that. （1） The NS3 genes of P-1,P-2 and P-3 cell strains decreased obviously than that of PK-15 cells（positive control） at the 72nd and 96th hour post-infection, respectively（P〈0.05） ,it showed the transcribed shRNA could interfere the reproduction of CSFV at RNA level. （2）The expressing quantity of NS3 protein of P-1,P-2 and P-3 cell strains were obvious less than that of PK-15 cells（positive control） at the 72nd and 96th hour post-infection, respectively（P〈0.05）, indicating that the transcribed shRNA could interfere the reproduction of CSFV at protein level, but the discrepancy were not obvious at the 148th hour post-infection.

关 键 词：半定量RT-PCR ELISA 猪瘟病毒 3′-UTR 

分 类 号：S852.651[农业科学—基础兽医学]