RESUMEN
Objective:To investigate whether coxsackievirus A 16 (CVA16) infection would affect the expression of N6-methyladenosine (m 6A) methylation-related proteins in human bronchial epithelial cells (16HBE), ICR suckling mice and SCRBA2 humanized mice and influence their subcellular localization. Methods:CVA16 was used to infect 16HBE cells at a multiplicity of infection (MOI) of 0.1 and mice at 10 7 CCID 50/ml. Changes in the expression of methyltransferases, demethylases and methylated reading proteins were analyzed by Western blot. Cellular localization of these proteins was observed using immunofluorescence. Results:The expression of m 6A methylation-related proteins was gradually reduced in CVA16-infected cells with time, but showed no obvious change in ICR suckling mice or SCRBA2 humanized mice. After infection, m 6A methylation-related proteins were redistributed in both the nucleus and cytoplasm and even degraded. Conclusions:CVA16 replication in host cells altered the expression and cellular localization of m 6A methylation-related proteins, which indicated that m 6A modification might be a new potential target for enterovirus therapy.
RESUMEN
We aimed to express and purify three rabies virus glycoproteins with different tags and sizes. After analyzing their binding function, we wish to obtain a rabies virus glycoprotein with higher affinity and ability to specifically bind memory B cells. Experiments were carried out to express full length, as well as the ectodomain RVG by gene engineering method. Combined with the antibody of CD19 and CD27, the candidate protein labeling with fluorescence was used to analyze its binding function. Flow cytometry was used to detect the anti-rabies virus specific memory B cells in PBMCs, and confirm the binding ability between the candidate proteins and anti-rabies virus-specific memory B cells. We successfully constructed three expression vectors pGEX-5X-1-RVG, pET28a-RVG and pET30a-G. Three glycoproteins GST-RVG, His-RVG and His-G were obtained by optimized expression and purification conditions. The antigen specificity of purified GST-RVG, His-RVG and His-G were identified by Western blotting and ELISA. The affinity of these three purified glycoproteins to anti-rabies virus antibody were detected by competitive ELISA. Anti-rabies virus specific memory B cells in positive PBMCs gained from people who had ever been injected with the vaccine can be detected by flow cytometry. Thus, we got a recombinant rabies virus glycoprotein that had high-affinity and could sort antigen specific memory B cells.