Preparation Of Polyclonal Antibodies Against The Novel Coronavirus Nsp1 Protein And Its Effect On The NF-κB/NLRP3 Signaling Pathwa

SARS-Co V-2 is a single-stranded positive-sense RNA enveloped virus that belongs to a member of the β coronavirus genus of the Coronaviridae family.Since it appeared in 2019,SARS-Co V-2 has led to hundreds of millions of infections and millions of deaths,which has caused huge impact on global public health,and economic and social development.The Nsp1 protein,as a key virulence factor of SARS-Co V-2 whose encoded gene is relatively conservative,has been confirmed in many studies to be an important target for vaccine,drug design and early detection.It is of great significance to build a viral detection platform based on Nsp1 and strengthen the functional research of Nsp1.Therefore,this study focused on the Nsp1 protein prepared mouse and rabbit polyclonal antibodies against the Nsp1 protein,respectively.Furthermore,the effect of Nsp1 protein on NF-κB/NLRP3 signaling was also investigated.The main research of this paper is performed as follows:(1)In this study,the recombinant plasmid p ET-28a-SARS-Co V-2-Nsp1 was constructed and transformed into E.coli BL21(DE3),and the recombinant Nsp1 protein with His tag was successfully expressed through optimizing expression conditions.After the cells were lysed,the solubility of Nsp1 was analyzed at the indicated time points.The results showed that Nsp1 protein was distributed in both the supernatant and precipitate,indicating that Nsp1 expressed in a soluble manner.Subsequently,the soluble protein distributed in the supernatant was harvested and the recombinant protein was purified with nickel-column affinity chromatography,followed by SDS-PAGE and Western blot identification.The results showed that the purified recombinant Nsp1 protein was successfully obtained.(2)BALB/c mice and New Zealand rabbits were immunized with purified recombinant Nsp1 protein emulsified with Freund adjuvant for three times,blood was collected and mouse and rabbit polyclonal antibodies were successfully prepared.In order to verify the effectiveness of mouse and rabbit polyclonal antibodies,the titers of polyclonal antibodies were measured by indirect ELISA method as purified protein served as coated antigen.The results showed that the titer of mouse polyclonal antibody was 1:800,and the titer of rabbit polyclonal antibody was1:320000.Subsequently,the specificity of polyclonal antibody was testified using Western blot,and the evidences revealed that recombinant Nsp1 protein could be probed by both mouse and rabbit polyclonal antibodies.(3)In order to further verify the effectiveness and specificity of both mouse and rabbit polyclonal antibodies,the eukaryotic expression vector p CMV-Myc-SARS-Co V-2-Nsp1 was constructed in this study.After Nsp1 protein was overexpressed,the effectiveness of polyclonal antibodies was detected by indirect immunofluorescence method.The results showed that Nsp1 protein was correctly expressed in cells,which was supported by that,compared with the control group,both mouse and rabbit polyclonal antibodies blotted with Nsp1 protein.These results indicated that the mouse and rabbit polyclonal antibodies against Nsp1 protein were successfully prepared in this study,both of which had good efficacy and specificity.(4)Several studies have reported that the uncontrolled inflammatory response caused by SARS-Co V-2 proteins might be crucial reason resulting in corona virus disease 2019(Covid-19),one of which is NF-κB signaling believed to play an important role in this process.In order to elucidate the influence of Nsp1 protein on NF-κB signaling,Western blot and RT-PCR techniques were used to determine the expression levels of p-p65,p65 and IκBα proteins,as well as IL-6,IL-8 and pro-IL-1β m RNA transcription levels,after overexpressing Nsp1 protein in cells.The results showed that Nsp1 degraded IκBα protein,up-regulated the expression levels of pp65/p65,and induced the m RNA transcription levels of IL-6,IL-8,and pro-IL-1β.These results suggest that Nsp1 can regulate the cytokines production through activating the NF-κB signaling that up-regulate the transcription levels of related inflammatory factors.(5)Pro-IL-1β is not only regulated by upstream NF-κB signaling,but also participates in the formation of downstream NLRP3 inflammasome(NLRP3,pro-caspase-1 and ASC)that activates caspase-1 to cleave IL-1β precursor to mature form.To further investigate the regulatory role of Nsp1 protein in pro-IL-1β cleavage triggered by the inflammasome,Western blot was used to detect the expression of pro-caspase-1,NLRP3,ASC and pro-IL-1β proteins after Nsp1 overexpression.The results showed that Nsp1 protein inhibited the expression of NLRP3,ASC and pro-IL-1β proteins,but exhibited no effect on the protein expression of pro-caspase-1.Next,the cleavage of pro-IL-1β protein was detected by Western blot,and the results showed inhibitory effect of Nsp1 protein on the cleavage of pro-IL-1β mediated by NLRP3,ASC and procaspase-1.These results suggest that Nsp1 protein inhibits the activation of NLRP3 inflammasome through down-regulating NLRP3 and ASC protein expression,thereby further inhibiting the cleavage of pro-IL-1β.At the same time,Nsp1 can also inhibit the release of inflammatory factors via inhibiting the expression of pro-IL-1β,which may be related to the mechanism ofNsp1 inhibiting host protein translation.In conclusion,both mouse and rabbit polyclonal antibodies against SARS-Co V-2 Nsp1 protein were successfully prepared,whose effectiveness and specificity were further verified in this study.In addition,the results showed that Nsp1 can enhance the transcription level of inflammatory factors in the NF-κB signaling.Besides,Nsp1 can down-regulate the protein expression of NLRP3-related signaling molecules to inhibit inflammatory signaling,which might be caused by the interaction that the inflammatory response was induced by Nsp1,but the host protein translation was blocked.This study can not only provide a platform foundation for developing diagnostic techniques for COVID-19,but also propose a new theoretical viewpoint for analyzing the function of Nsp1 and the inflammatory response caused by SARS-Co V-2.