Inhibitory Effects of Porcine Milk-derived Exosome on Porcine Epidemic Diarrhea Virus

doi:10.13560/j.cnki.biotech.bull.1985.2021-0112

Abstract

Abstract:

This work aims to explore the effects of porcine milk-derived exosome on porcine epidemic diarrhea virus in IPEC-J2 cells. We adapted crystal violet and MTT methods to measure cytoactive,and real-time-PCR and Western Blot to measure the expressions of PEDV virus and cell relative genes and proteins,respectively. The results showed that the porcine milk-derived exosome significantly inhibited the infectivity of PEDV virus to IPEC-J2 cells,and the cell survival rate and activity significantly increased（P<0.05）. The M,N,ORF3,Spike,RNA polymerase and E genes of the virus inside the infected cells with PEDV and cell supernatant were extremely down-regulated（P<0.01）. The PEDV virus N protein and IPEC-J2 cells’ apoptotic CLDN1 protein expressions were significantly inhibited by the porcine milk-derived exosome. Under different treatments,treatment 2（kill）and 3（repair）had no effect on the expressions of immune-related IFN-a and IRF3 genes respectively（P>0.05）,and the other treatments extremely down-regulated the expression of pAPN（P<0.01）,but had no significant effect on the expression of NF-κB gene（P>0.05）. All above results suggest that porcine milk-derived exosome under 3 different treatments may reduce the infectivity of the PEDV to the intestinal epithelial IPEC-J2 cells in piglets. It is speculated that its mechanism may reduce infectivity by inhibiting viral infection or replication-related genes on the one hand,and on the other hand,or enhance the resistance of cells to viruses by reducing intracellular apoptosis or increasing the expressions of immune-related genes t,so as to achieve the protective effect of piglet intestinal epithelial cells,but the specific regulation mechanism needs to be further investigated.

Key words: porcine milk-derived exosome, porcine epidemic diarrhea virus, inhibitory effect

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.

Figures/Tables 8

References 36

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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

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