Co-expression and antiviral activity analysis of three human host restriction fac-tors CH25H-IFITM3-ISG15
10.16303/j.cnki.1005-4545.2025.01.08
- VernacularTitle:3种人源宿主限制因子CH25H-IFITM3-ISG15的共表达及抗病毒活性分析
- Author:
Rong JIANG
1
;
Letian LI
;
Chunmei CUI
;
Chang LI
Author Information
1. 广西大学动物科学技术学院,广西南宁 530005;中国农业科学院长春兽医研究所,吉林长春 130122
- Publication Type:Journal Article
- Keywords:
host restriction factors;
CH25H;
IFITM3;
ISG15;
antiviral activity
- From:
Chinese Journal of Veterinary Science
2025;45(1):53-58
- CountryChina
- Language:Chinese
-
Abstract:
To explore the application effect of host restriction factors(HRFs)in the development of antiviral gene drugs,this study based on the"common pathway of viral infection"and"host innate immunity HRFs",the genes of three antiviral HRFs,namely cholesterol-25-hydroxylase(CH25H),interferon-induced transmembrane protein(IFITM3)and interferon-stimulated gene 15(ISG15)were fused and expressed using pCK vectors to construct antiviral gene drugs.The fusion expression gene sequence CH25H-IFITM3-ISG15(C Ⅱ)was constructed by linking the gene cod-ing sequences of these three HRFs through the cleavage peptide coding sequence.Subsequently,the C Ⅱ sequence was amplified by PCR,ligated to the pCK expression vector,and the recombinant plasmid was transformed,identified,and extracted to obtain a candidate biodrug based on the DNA expression system,which was named pCK-CⅡ.Then,the recombinant plasmid was transfected in-to HEK 293T cells,and the expression of three antiviral proteins was successfully detected by Western blot.To clarify the antiviral effect of pCK-C Ⅱ at the cellular level,pCK-C Ⅱ was trans-fected into HEK 293 cells and BHK-21 cells,respectively.Twenty-four hours later,the BHK-21 cells were infected with vesicular stomatitis virus expressing green fluorescent protein(VSV-GFP),12 h later,the cells were observed under a fluorescence microscope and detected by flow cy-tometry;HEK 293 cells were inoculated with H3N2 subtype influenza virus,and the expression of H3N2 subtype influenza virus nuclear proteins was detected after 12 h using Western blot.The re-sults showed that transient transfection of pCK-C Ⅱ plasmid could significantly reduce the fluores-cence level of cells and the expression of nuclear protein of H3N2 subtype influenza virus in infec-ted cells.These results indicated that pCK-C Ⅱ had an inhibitory effect on the infection of VSV-GFP and H3N2 subtypes of influenza viruses after transient transfection of cells.
