携带CRISPR/Cas9的重组腺联病毒对伪狂犬病病毒感染小鼠的治疗效应

doi:10.11843/j.issn.0366-6964.2022.03.016

摘要/Abstract

摘要： 猪伪狂犬病病毒(pseudorabies virus,PRV)是猪伪狂犬病的病原。目前,针对该病毒有较多成熟的商品化疫苗,但病毒变异频繁,因而在生猪养殖中伪狂犬病的发生仍然较为普遍。如何清除宿主体内野毒是防控该病的关键所在。应用腺联病毒携载CRISPR/Cas9系统,以小鼠为动物模型,针对PRV的TK基因、gE基因和VP16基因进行剪切,减弱PRV毒力和复制能力,以期清除小鼠体内的PRV。结果表明,成功构建带有CRISPR/Cas9表达系统及相应sgRNA的pX601重组质粒(pXTK、pXGE、pXVP16);并包装成具有基因编辑功能的AAV-gE、AAV-VP16和AAV-TK病毒;经细胞水平和小鼠的感染试验,证实该方法能明显降低细胞和小鼠体内的PRV载量,并且延长小鼠存活时间,提高小鼠成活率,该治疗结果与治疗次数呈正相关。该小鼠模型的研究结果为猪伪狂犬病的临床治疗提供了新的借鉴。

关键词: 猪伪狂犬病病毒, CRISPR/Cas9, 腺联病毒, 治疗

Abstract: Pseudorabies virus (PRV) is the pathogen of pig pseudorabies. At present, there are more mature commercial vaccines in the clinical application, but the virus variation is frequent, so the occurrence of pseudorabies is still more common in pig breeding. How to clean the wild virus in the host is the key to the prevention and control of the disease. The adeno-associated virus (AAV) carrying CRISPR/Cas9 system toward TK, gE and VP16 genes of PRV were designed to weaken the virulence and replication ability of PRV, so as to achieve the purpose of eliminating PRV in mice model. The results showed that the pX601 recombinant plasmids (pXTK, pXGE, pXVP16) with CRISPR/Cas9 expression system and corresponding sgRNAs were successfully constructed, and these plasmids were further packaged into AAV-gE, AAV-VP16 and AAV-TK viruses with gene editing function, which significantly reduced the PRV load in cells and mice and increased the survival rate in the cell level and mouse infection test. The treatment result is positively correlated with the times of treatments. The results of the mouse model provide a new reference for the clinical treatment of porcine pseudorabies.

Key words: pseudorabies virus, CRISPR/Cas9 system, adeno-associated virus, therapy

中图分类号:

S852.659.1

李兆龙, 张惠芳, 丰志华, 方舟. 携带CRISPR/Cas9的重组腺联病毒对伪狂犬病病毒感染小鼠的治疗效应[J]. 畜牧兽医学报, 2022, 53(3): 834-846.

LI Zhaolong, ZHANG Huifang, FENG Zhihua, FANG Zhou. Therapeutic Effect of Recombinant Adeno-Associated Virus Carrying CRISPR/Cas9 on Pseudorabies Virus-infected Mice[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(3): 834-846.

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