微生物學(xué)報 Acta Microbiologica Sinica
48(11)：1507~1513; 4 November 2008
ISSN 0001-6209; CN11-1995/Q
載體表達(dá)的siRNA分子對豬圓環(huán)病毒2型復(fù)制的抑制作用
王海燕,，劉文博，高崧*,，劉秀梵
(揚州大學(xué)農(nóng)業(yè)部畜禽傳染病學(xué)重點開放實驗室,，揚州 225009)
摘要：【目的】尋找一種基于RNA干擾技術(shù)的豬圓環(huán)病毒2型感染的防控方法?！痉椒ā扛鶕?jù)豬圓環(huán)病毒2型毒株基因組核苷酸序列,，設(shè)計了3條特異性小干擾RNA（short interfering RNA,，siRNA）分子，其中2條針對豬圓環(huán)病毒1型和2型復(fù)制酶基因（rep）,，1條針對豬圓環(huán)病毒2型核衣殼蛋白基因(cap),，將合成的DNA片段退火形成雙鏈，分別連接到RNAi-Ready pSIREN-RetroQ ZsGreen 載體鼠源U6啟動子下游,，轉(zhuǎn)化大腸桿菌得到陽性克隆,，測序鑒定后分別命名為Retro-SH1，Retro-SH4,，Retro-SH6,。用上述質(zhì)粒轉(zhuǎn)染PCV2感染前,、后的Dulac細(xì)胞及肌肉注射PCV2感染前、后的BALB/c小鼠,，應(yīng)用實時定量PCR試驗評價其對病毒在細(xì)胞及小鼠體內(nèi)復(fù)制的抑制作用,，免疫組化法檢測脾臟中病毒的存在?！窘Y(jié)果】感染PCV2前或后轉(zhuǎn)染500 ng Retro-SH1,，Retro-SH4,，Retro-SH6質(zhì)粒能有效抑制PCV2在Dulac細(xì)胞上的復(fù)制，抑制率最高可達(dá)99%以上,，對10株不同來源的臨床分離株在細(xì)胞中復(fù)制的抑制作用同樣明顯,，且不同毒株間差異不大。動物試驗中,，肌肉注射10 g上述不同siRNA分子對小鼠體內(nèi)PCV2的復(fù)制有一定的抑制作用,，其抑制率在26%至99%之間?！窘Y(jié)論】載體表達(dá)的siRNA分子可能成為防控豬圓環(huán)病毒2型感染的一種新工具,。
關(guān)鍵詞：RNA干擾,；豬圓環(huán)病毒2型；復(fù)制,；抑制
中圖分類號：R392 文獻(xiàn)標(biāo)識碼：A 文章編號：0001-6209 (2008) 11-1507-07
Inhibition of porcine circovirus type 2 replication by the plasmid vector expressing siRNAs
Haiyan Wang, Wenbo Liu, Song Gao*, Xiufan Liu
(Key Laboratory of Animal Infectious Diseases of Ministry of Agriculture, Yangzhou University, Yangzhou 225009, China)
Abstract: [Objective] To develop siRNA as a potential tool for control of porcine circovirus 2 (PCV2) infection. [Methods] We designed two short interfering RNAs (siRNAs) related to the replicase (rep) gene of porcine circovirus and one related to capsid (cap) gene of PCV2 basd on the genomic sequences of the viruses deposited in GenBank. The corresponding DNA fragments were synthesized, annealed and ligated into the downstream of the mouse originated U6 promoter of the RNAi-Ready pSIREN-RetroQ ZsGreen vector. As controls, the siRNAs specific to Luciferase contained in the vector kit and negative fragments with no similarities to any known species were also included. We transformed the recombinant plasmids into the host bacterium DH5α and positive clones were selected. The positive clones were sequenced and five clones carried the correct inserts. They were designated as Retro-SH1,，Retro-SH4,，Retro-SH6,，Retro-Luc and Retro-NC. To test whether the siRNAs specific to PCV expressed by the vector could inhibit the virus replication, we evaluated the inhibition effect on PCV2 replication both in Dulac cells and experimental mice using real-time PCR and immunohistochemistry. [Results] The results showed that the purified plasmids could strongly inhibit the replication of the PCV2 virus and the inhibition rate reached to 99% in cell culture. In the animal experiments, siRNAs expressed by the plasmids could inhibit the replication of the PCV2 virus and the inhibition rate varied from 26% to 99%. [Conclusion] The siRNAs specific to PCV2 expressed by vectors should be potential in the control of the diseases related to PCV2 infection.
Keywords: RNA interference; porcine circovirus type 2; replication; inhibition
生物谷推薦全文下載：載體表達(dá)的siRNA分子對豬圓環(huán)病毒2型復(fù)制的抑制作用
更多全文請查看鏈接：http://journals.im.ac.cn
聲明：本文由《微生物學(xué)報》授權(quán)生物谷 www.bioon.com 網(wǎng)站發(fā)布，如需轉(zhuǎn)載請直接與中國科學(xué)院微生物研究所期刊聯(lián)合編輯部聯(lián)系并支付相應(yīng)費用,，未經(jīng)授權(quán)不得轉(zhuǎn)載,，若轉(zhuǎn)載將付相應(yīng)的法律責(zé)任
