氨基肽酶（<i>pAPN</i>）和唾液神经氨酸酶（<i>NEU</i>3）敲除对TGEV病毒侵染的影响

李兆龙; 丰志华; 张冰晨; 方舟; 梁旺旺; 陈文志

氨基肽酶（pAPN）和唾液神经氨酸酶（NEU3）敲除对TGEV病毒侵染的影响

Role of pAPN and NEU3 in TGEV Infection on Pig

摘要

摘要:
目的氨基肽酶（ pAPN ）是猪的传染性胃肠炎病毒（TGEV）侵染的主要受体，验证pAPN和唾液酸神经氨酸酶（NEU3）双基因对猪的传染性胃肠炎病毒（TGEV）入侵机制的影响。
方法应用CRISPR-Cas9基因编辑技术，敲除猪睾丸细胞（ST）的pAPN和NEU3两个基因。经过病毒感染试验，测定敲除pAPN和NEU3两个目标基因的ST细胞对病毒的侵染变化以及病毒拷贝数的变化、细胞病变改善，同时监测病毒侵染后纤连蛋白的变化。
结果与对照组相比，敲除pAPN和NEU3两个目标基因的ST细胞，明显减轻TGEV侵染引起的细胞病变，TGEV的拷贝数也出现明显下降。此外，同样滴度的TGEV侵染pAPN和NEU3双基因敲除ST细胞后，诱导ST细胞的免疫应答物IFNβ的mRNA水平明显低于野生型ST细胞组。
结论 pAPN和NEU3双基因的敲除，明显降低了ST细胞中的TGEV病毒拷贝数，同时也减少病毒引起的细胞病变，这个结果证实细胞中的pAPN和NEU3双基因可作为将来养猪生产中抗病毒治疗及抗病品种选育的靶基因。

Abstract:
Objective Role of aminopeptidase gene pAPN and sialic acid neuraminidase gene NEU3 in the transmissible gastroenteritis virus (TGEV) infection on pigs was investigated.
Methods Being the main receptor of TGEV, pAPN was removed from pAPN and NEU3 in ST cells to verify its supposed key function on the disease. The CRISPR gene editing technique was applied to clip the target gene in ST cells prior to an artificial TGEV infection test. The resulting changes on the infection, virus copy number, cytopathic improvement, and fibronectin were monitored.
Results Compared with control, the ST cells free of pAPN and NEU3 significantly attenuated TGEV infection-induced cytopathies and the virus copy number. In addition, at a same TGEV titer the mRNA immune responders induced by the knockdown ST cells were significantly lower than the wild-type counterparts.
Conclusion It was confirmed that the removal of pAPN and NEU3 inhibited the TGEV infection in pigs with reduced viral induced cytopathies. Thus, an antiviral therapy and a guideline for breeding resistant pigs could be developed by targeting these two key genes in the ST cells.

HTML全文

参考文献(22)

施引文献

资源附件(0)