Preparation of polyclonal antibodies against Scylla paramamosain STAT and its application in immune responses

Abstract

Signal transducers and activators of transcription (STAT), as a core component of the Janus kinase (JAK)/STAT signaling pathway, play a vital regulatory role in the innate antiviral immunity and immune signal transduction in Scylla paramamosain . To enable comprehensive biological studies, we developed and validated a specific polyclonal antibody against this target. In this study, the properties of STAT protein were characterized using bioinformatics, and the full-length STAT coding sequence was then cloned into the recombinant plasmid pET-32a-STAT for expression in Escherichia coli. The recombinant STAT protein, which was induced and collected from the gel, was employed to immunize mice, resulting in the production of specific polyclonal antibodies. The antibody was then used to investigate STAT protein expression. Our findings confirmed that the STAT protein was successfully produced in a prokaryotic system. The generated mouse anti STAT polyclonal antibody exhibited a high titer of 1:64,000 and specifically recognized STAT expressed in both prokaryotic and eukaryotic systems. Furthermore, the anti-STAT polyclonal antibody specifically recognized endogenous STAT in various Scylla paramamosain tissues and revealed a significant nuclear translocation of SpSTAT in hemocytes following poly(I:C) stimulation. With its high specificity and titer, this antibody represents a useful reagent for studying the role of STAT in antiviral innate immunity and immune signaling in Scylla paramamosain .