干扰素诱导跨膜蛋白1对猪源冠状病毒吸附宿主细胞的影响

doi:10.16431/j.cnki.1671-7236.2021.04.024

Abstract

Abstract: In order to explore the effect of interferon-inducible transmembrane protein 1 (IFITM1) on the replication of porcine coronaviruses,the highly pathogenic virus strain of Transmissible gastroenteritis virus (TGEV) and its host cell PK15 were selected as the research objects.After normal PK15 cells were inoculated with TGEV,Real-time PCR was used to detect the mRNA expression levels of various interferon-stimulated genes (ISGs) in PK15 cells at different time points post infection.PK15 cell lines that stably expressed and stably interfered with the expression of IFITM1 were constructed through the lentiviral expression system.A shRNA sequence targeted to interfere with porcine IFITM1 and a full-length porcine IFITM1 sequence were inserted into pLKO.1-EGFP-Puro vector and pLVML-Myc-MCS-IRES-Puro vector to construct recombinant plasmids pLKO.1-IFITM1shRNA-EGFP-Puro and pLVML-Myc-IFITM1-IRES-Puro,respectively.The recombinant plasmids and the lentiviral packaging plasmids were co-transfected into 293FT cells to obtain a recombinant lentivirus with the target gene.After the lentivirus infected PK15 cells,it was screened with puromycin to obtain a PK15 cell line that stably expressed and stably interfered with the expression of IFITM1,named PK15-IFITM1 and PK15-IFITM1^-/-,respectively,and Real-time PCR,IFA and Western blotting were used to detect the interference efficiency and expression of IFITM1.TGEV was inoculated with PK15-IFITM1^-/- and PK15-IFITM1,and Real-time PCR was used to determine the copies of TGEV in the cells.The results showed that the mRNA level of some ISGs increased within 48 h after PK15 cells were inoculated with TGEV.The interference efficiency of PK15-IFITM1^-/-cell line was 70%,and the expression of PK15-IFITM1 cell line was successful.In the PK15-IFITM1^-/- cell line,the mRNA level of IFITM1 was significantly down-regulated,which promoted the replication of TGEV.Conversely,in the PK15-IFITM1 cell line,the replication of TGEV was inhibited.However,the expression or absence of IFITM1 did not affect the adsorption of TGEV to PK15.In short,IFITM1 had a significant antiviral effect on TGEV,IFITM1 had no effect on the adsorption of TGEV to PK15 cells,which laid the foundation for subsequent research on the anti-coronavirus mechanism of IFITM1.

Key words: interferon-inducible transmembrane protein 1 (IFITM1); coronavirus; Transmissible gastroenteritis virus (TGEV)

CLC Number:

S852.4

YAN Kexin, DENG Zhibang, HE Jiayi, YUAN Qichao, WANG Jiaoshun, YUAN Xiaomin. Effect of Interferon-inducible Transmembrane Protein 1 on the Adsorption of Porcine Coronavirus to Host Cells[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(4): 1370-1380.

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Effect of Interferon-inducible Transmembrane Protein 1 on the Adsorption of Porcine Coronavirus to Host Cells

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