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

为探索干扰素诱导跨膜蛋白1（interferon-inducible transmembrane protein 1，IFITM1）对猪源冠状病毒复制的影响，本研究选用猪传染性胃肠炎病毒（Transmissible gastroenteritis virus，TGEV）高致病毒株及其宿主细胞PK15作为研究对象。正常PK15细胞接种TGEV后，实时荧光定量PCR检测感染后不同时间点PK15细胞中一些干扰素刺激基因（interferon-stimulated genes，ISGs）的mRNA表达水平；利用慢病毒表达系统构建稳定表达和稳定干扰IFITM1表达的PK15细胞系。将一段靶向干扰猪源IFITM1的shRNA序列及猪源IFITM1全长，分别插入pLKO.1-EGFP-Puro载体及pLVML-Myc-MCS-IRES-Puro载体中，分别构建出pLKO.1-IFITM1shRNA-EGFP-Puro及pLVML-Myc-IFITM1-IRES-Puro重组质粒。将重组质粒与慢病毒包装质粒共转染293FT细胞后获得带有目的基因的重组慢病毒，慢病毒侵染PK15细胞后用嘌呤霉素进行筛选，获得稳定表达及稳定干扰IFITM1表达的PK15细胞系，分别命名为PK15-IFITM1及PK15-IFITM1^-/-，并分别用实时荧光定量PCR、间接免疫荧光试验（IFA）及Western blotting检测IFITM1干扰效率及表达情况；TGEV接种PK15-IFITM1^-/-和PK15-IFITM1，实时荧光定量PCR测定细胞中TGEV的拷贝数。结果显示，PK15细胞接种TGEV后的48 h内，一些ISGs的mRNA水平均有所上升；PK15-IFITM1^-/-细胞系的干扰效率为70%，PK15-IFITM1细胞系表达成功；在PK15-IFITM1^-/-细胞系中，IFITM1的mRNA水平显著下调，促进了TGEV的复制。反之，在PK15-IFITM1细胞系中，TGEV的复制受到了抑制。但IFITM1的表达或缺失却不影响TGEV对PK15的吸附作用。总之，IFITM1对TGEV有显著的抗病毒作用，IFITM1不影响TGEV对PK15细胞的早期吸附，这为后续IFITM1抗冠状病毒机制的研究奠定了基础。

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

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.