生物技术通报 ›› 2021, Vol. 37 ›› Issue (12): 141-150.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0112
陈婷1,2(), 谢梅英3, 魏立民1, 欧阳坤2, 程晓1(), 张永亮2()
收稿日期:
2021-01-28
出版日期:
2021-12-26
发布日期:
2022-01-19
作者简介:
陈婷,女,博士,研究方向:动物分子营养;E-mail: 基金资助:
CHEN Ting1,2(), XIE Mei-ying3, WEI Li-min1, OUYANG Kun2, CHENG Xiao1(), ZHANG Yong-liang2()
Received:
2021-01-28
Published:
2021-12-26
Online:
2022-01-19
摘要:
本文旨在探索猪乳外泌体(exosome)对仔猪小肠上皮细胞(IPEC-J2)中PEDV的抑制作用。试验采用结晶紫染色及MTT方法分别测定细胞活性,qRT-PCR和Western blot分别测定病毒及细胞相关基因和蛋白的表达水平。结果显示,猪乳exosome显著抑制PEDV病毒对IPEC-J2细胞的感染力,细胞存活率和活性显著升高(P<0.05);极显著下调感染PEDV后细胞内及细胞上清中病毒的M、N、ORF3、Spike、RNA polymerase和E等基因表达量(P<0.01);猪乳exosome组细胞内PEDV病毒N蛋白及IPEC-J2细胞内凋亡CLDN1 蛋白表达量显著下调;猪乳exosome三种不同处理方式下,处理方式2(杀灭)和3(修复)分别对免疫相关IFN-a和IRF3基因表达影响不明显(P>0.05),其他各处理组对猪氨肽酶N(pAPN)表达量极显著下调(P<0.01),而对NF-κB基因的表达无显著影响(P>0.05);以上结果提示,猪乳exosome在3种不同的处理方式下均能降低PEDV对仔猪肠道上皮IPEC-J2细胞的感染能力,推测其机制可能一方面通过抑制病毒感染或复制相关的基因降低感染性,另一方面通过降低细胞内凋亡或提高免疫相关基因的表达增强细胞对病毒的抵抗能力,从而达到对仔猪肠道上皮细胞的保护作用。
陈婷, 谢梅英, 魏立民, 欧阳坤, 程晓, 张永亮. 猪乳外泌体对猪流行性腹泻病毒的抑制作用[J]. 生物技术通报, 2021, 37(12): 141-150.
CHEN Ting, XIE Mei-ying, WEI Li-min, OUYANG Kun, CHENG Xiao, ZHANG Yong-liang. Inhibitory Effects of Porcine Milk-derived Exosome on Porcine Epidemic Diarrhea Virus[J]. Biotechnology Bulletin, 2021, 37(12): 141-150.
Gene | Primer sequence(5'-3') |
---|---|
ORF3-F | CGGGCTTCGTTTAGTCTGCT |
ORF3-R | GATGTAATGGTCGCCACCTTCT |
N protein-F | AAAACGGGTGCCATTATCTCT |
N protein-R | CCATTTGCTGGTCCTTATTCC |
M protein-F | TCCCGTTGATGAGGTGATTG |
M protein-R | AAGGATGCTGAAAGCGAAAA |
RNA polymerase-F | GACCGCAGGCTATCCTTTGA |
RNA polymerase-R | GCTCTATCGCACTTTGGGTAATC |
spike protein-F | GATGACATTTATTCCCGACTGG |
spike polymerase-R | GCTGAGATTGCGATTTGACG |
S protein-F | GCAGTAATTCCTCAGATCCTC |
S protein-R | GTAGTGTCAGATGCAATGAGG |
E protein-F | GGCTCAGAGCAAGAGAGGTATCC |
E protein-R | GGTCTCAAACATGATCTGAGTCATCT |
NF-Kb-F | CCAGCCCTATCCCTTTACGC |
NF-Kb-R | GCCTCTGTCAGTGTCCCTTCC |
IFN-F | GCAACCAGGTCCAGAAGGC |
IFN-R | GACCTAGTCGTCGAGTCCC |
IRF-3-F | GCACTCACCGTCGTCATTC |
IRF-3-R | CAGAAAAGGCCGTGGAAATA |
p-APN-F | GGATTGTTCCCATCTCATCTATT |
p-APN-R | TTTTGGCGTAGCCTGCT |
表1 引物序列
Table 1 Primer sequences
Gene | Primer sequence(5'-3') |
---|---|
ORF3-F | CGGGCTTCGTTTAGTCTGCT |
ORF3-R | GATGTAATGGTCGCCACCTTCT |
N protein-F | AAAACGGGTGCCATTATCTCT |
N protein-R | CCATTTGCTGGTCCTTATTCC |
M protein-F | TCCCGTTGATGAGGTGATTG |
M protein-R | AAGGATGCTGAAAGCGAAAA |
RNA polymerase-F | GACCGCAGGCTATCCTTTGA |
RNA polymerase-R | GCTCTATCGCACTTTGGGTAATC |
spike protein-F | GATGACATTTATTCCCGACTGG |
spike polymerase-R | GCTGAGATTGCGATTTGACG |
S protein-F | GCAGTAATTCCTCAGATCCTC |
S protein-R | GTAGTGTCAGATGCAATGAGG |
E protein-F | GGCTCAGAGCAAGAGAGGTATCC |
E protein-R | GGTCTCAAACATGATCTGAGTCATCT |
NF-Kb-F | CCAGCCCTATCCCTTTACGC |
NF-Kb-R | GCCTCTGTCAGTGTCCCTTCC |
IFN-F | GCAACCAGGTCCAGAAGGC |
IFN-R | GACCTAGTCGTCGAGTCCC |
IRF-3-F | GCACTCACCGTCGTCATTC |
IRF-3-R | CAGAAAAGGCCGTGGAAATA |
p-APN-F | GGATTGTTCCCATCTCATCTATT |
p-APN-R | TTTTGGCGTAGCCTGCT |
图5 猪乳exosome对PEDV相关基因在IPEC-J2细胞上清中表达的影响
Fig. 5 Expressions of PEDV-related genes treated with porcine milk-derived exosome in IPEC-J2 cells supernatant
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