生物技术通报 ›› 2022, Vol. 38 ›› Issue (11): 90-96.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0087
钟菁(), 孙玲玲, 张姝, 蒙园, 支怡飞, 涂黎晴, 徐天鹏, 濮黎萍, 陆阳清()
收稿日期:
2022-01-19
出版日期:
2022-11-26
发布日期:
2022-12-01
作者简介:
钟菁,女,硕士研究生,研究方向:动物繁殖新技术;E-mail:基金资助:
ZHONG Jing(), SUN Ling-ling, ZHANG Shu, MENG Yuan, ZHI Yi-fei, TU Li-qing, XU Tian-peng, PU Li-ping, LU Yang-qing()
Received:
2022-01-19
Published:
2022-11-26
Online:
2022-12-01
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。
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.
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 |
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 |
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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