应用CRISPR/Cas9技术敲除Mda5基因对新城疫及传染性法氏囊病毒复制的影响

doi:10.13560/j.cnki.biotech.bull.1985.2022-0087

摘要/Abstract

摘要：

采用CRISPR/Cas9技术敲除DF-1的黑色素瘤分化相关基因5（melanoma-differentiation-associated gene 5，Mda5），探索Mda5基因对新城疫病毒（Newcastle disease virus，NDV）和传染性法氏囊病毒（infectious bursal disease virus，IBDV）复制的影响。通过CRISPR/Cas9技术构建Mda5敲除的DF-1细胞系；实时荧光定量PCR和免疫荧光法检测病毒感染Mda5敲除细胞后病毒RNA水平、细胞病变情况及抗病毒相关基因mRNA水平等。获得Mda5基因敲除的DF-1细胞；与对照组细胞相比，细胞形态、贴壁能力和增殖能力均无显著性差异；与对照组细胞相比，Mda5基因敲除的DF-1细胞感染IBDV后，IFN-β和PKR表达显著性下调，CH25H的表达和病毒复制水平均无显著性差异；感染NDV后，IFN-β表达和病毒复制水平显著性下调，CH25H表达量显著性上调，PKR表达无显著性差异。CRISPR/Cas9技术建立Mda5敲除的DF-1中，IFN-β虽然显著应答IBDV和NDV感染，但不能显著抑制病毒的复制，说明Mda5并非抗病毒复制的关键模式识别受体。

关键词: Mda5, 新城疫病毒, 传染性法氏囊病毒, CRISPR/Cas9, 基因敲除

Abstract:

The melanoma-differentiation-associated gene 5（Mda5）of DF-1 was knocked down using CRISPR/Cas9 technology to explore the effect of the Mda5 gene on the replication of Newcastle disease virus（NDV）and infectious bursal disease virus（IBDV）replication. CRISPR/Cas9 gene editing technology was applied to construct Mda5 knockout DF-1 cell lines，and real-time fluorescence quantitative PCR and immunofluorescence were used to detect the RNA levels of viruses，cytopathic conditions and mRNA levels of antiviral-related genes after IBDV and NDV infection of Mda5 knockout cells. Mda5 knockout DF-1 cells were obtained；there were no significant differences in cell morphology，apposition ability and proliferation ability compared to control cells. Compared to control cells，Mda5 knockout DF-1 cells infected with IBDV showed significant down-regulation of IFN-β and PKR expression，no significant differences in CH25H expression and viral replication levels. After infection with NDV，IFN-β expression and viral replication levels were significantly down-regulated，CH25H expression was significantly up-regulated，and PKR expression was not significantly different after infection with NDV. In DF-1 while applying CRISPR/Cas9 technology to knockout Mda5，though IFN-β significantly responded to IBDV and NDV infection，failed to significantly inhibit viral replication，suggesting that Mda5 is not a key pattern recognition receptor for antiviral replication.

Key words: Mda5, Newcastle disease virus, infectious bursa disease virus, CRISPR/Cas9, knockout

钟菁, 孙玲玲, 张姝, 蒙园, 支怡飞, 涂黎晴, 徐天鹏, 濮黎萍, 陆阳清. 应用CRISPR/Cas9技术敲除Mda5基因对新城疫及传染性法氏囊病毒复制的影响[J]. 生物技术通报, 2022, 38(11): 90-96.

ZHONG Jing, SUN Ling-ling, ZHANG Shu, MENG Yuan, ZHI Yi-fei, TU Li-qing, XU Tian-peng, PU Li-ping, LU Yang-qing. Effect of Knocking Out the Mda5 Gene by CRISPR/Cas9 Technology on the Replication of Newcastle Disease and Infectious Bursal Virus[J]. Biotechnology Bulletin, 2022, 38(11): 90-96.

图/表 7

表1 sgRNA1-6引物序列

Table 1 sgRNA1-6 primer sequences

引物名称Primer name	序列Sequence（5'-3'）
sg RNA1	F：CACCGTGTAGAGGAAGCGCTCGTCT R：AAACAGACGAGCGCTTCCTCTACAC
sg RNA2	F：CACCGCTGCTATGCGCCGTGGAACG R：AAACCGTTCCACGGCGCATAGCAGC
sg RNA3	F：CACCGTCGCGGCGGCCACGTTCCA
	R：AAACTGGAACGTGGCCGCCGCGAC
sg RNA4	F：CACCGTGAACCATCCGGGGTCGCGG R：AAACCCGCGACCCCGGATGGTTCAC
sg RNA5	F：CACCgCAGTGAACCATCCGGGGTCG R：AAACCGACCCCGGATGGTTCACTGC
sg RNA6	F：CACCGCTGGGGTTCACGTAGCAAG R：AAACCTTGCTACGTGAACCCCAGC

表2 qPCR引物序列

Table 2 qPCR primer sequences

引物名称Primer name	序列Sequence（5'-3'）
IFN-β qPCR F	TGCAACCATCTTCGTCACCA
IFN-β qPCR R	GGAGGTGGAGCCGTATTCTG
PKR qPCR F	CCTATGCAATCAAACGAGTTGAG
PKR qPCR R	GTCCCTTCCCAGCTGCAATA
qPCR（CH25H）F qPCR（CH25H）R	ATCCATTCCTCCTCGGATGC AAAGGCACAAGTCGGTGAGT

图1 重组质粒构建结果 A：sgRNA骨架；B：sgRNA expression vector测序骨架

Fig. 1 Results of recombinant plasmid construction A：sgRNA skeleton；B：sgRNA expression vector sequencing framework

图2 MDA5 KO细胞系建立 A：MDA5 KO转染后24 h；B：MDA5 KO细胞PCR鉴定结果图；C：MDA5 KO细胞PCR产物测序结果图

Fig. 2 Establishment of MDA5 KO cell line A：24 h after MDA5 KO transfection；B：PCR identification results graph of MDA5 KO cells；C：sequencing results of PCR products from MDA5 KO cells

图3 MDA5 KO与普通DF-1形态及生长速度比较 A：普通DF-1和MDA5 KO解冻后24 h；B：普通DF-1和MDA5 KO解冻后72 h；C：普通DF-1和MDA5 KO解冻后24 h计数结果；D：普通DF-1和MDA5 KO解冻后1-3 d生长曲线

Fig. 3 Comparison of morphology and growth rate between MDA5 KO and ordinary DF-1 A：24 h after conventional DF-1 and MDA5 KO were thawed；B：72 h after conventional DF-1 and MDA5 KO were thawed；C：count results of conventional DF-1 and MDA5 KO at 24 h after thawing；D：growth curves of ordinary DF-1 and MDA5 KO 1-3 d after thawing

图4 IBDV感染MDA5 KO后结果 A：普通DF-1感染IBDV前后对比；B：MDA5 KO感染IBDV前后对比；C：IBDV感染后干扰素IFN-β基因检测结果；D：IBDV感染后PKR基因检测结果；E：IBDV感染后CH25H基因检测结果；F：IBDV感染后IBDV基因检测结果

Fig. 4 Results of IBDV infection with MDA5 KO A：Comparison of normal DF-1 before and after infection with IBDV.B：Comparison of MDA5 KO before and after infection with IBDV. C：Results of IFN-β gene detection after IBDV infection. D：PKR gene test results after IBDV infection. E：Results of CH25H gene detection after IBDV infection. F：Results of IBDV gene detection after IBDV infection

图5 NDV感染MDA5 KO后结果 A：普通DF-1感染NDV前后对比；B：MDA5 KO感染NDV前后对比；C：NDV感染后干扰素IFN-β基因检测结果；D：NDV感染后PKR基因检测结果；E：NDV感染后CH25H基因检测结果；F：NDV感染后NDV基因检测结果

Fig. 5 Results of NDV infection with MDA5 KO A：Comparison of normal DF-1 before and after infection with NDV. B：Comparison of MDA5 KO before and after infection with NDV. C：Results of IFN-β gene detection after NDV infection. D：PKR gene test results after NDV infection. E：Results of CH25H gene detection after NDV infection. F：Results of NDV gene detection after NDV infection

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