DOI: 10.57760/sciencedb.j00217.07379
title: Bioinformatics Analysis, Prokaryotic Expression, and Antiserum Preparation of hnRNP A1 Protein
title: hnRNP A1蛋白生物信息学分析、原核表达及多克隆制备

description: Objective This study aims to construct a recombinant expression plasmid using methods such as PCR and double enzyme digestion. By conducting single-factor experiments to adjust IPTG concentration, expression time, and expression temperature, the research seeks to optimize the expression conditions of the hnRNP A1 protein in BL21 competent cells. The goal is to obtain high-concentration, high-quality purified protein and to prepare high-titer polyclonal antibodies against hnRNP A1.Methods Bioinformatics tools were used to analyze the physicochemical properties and structure of hnRNP A1. The pET-28a-hnRNP A1 recombinant plasmid was constructed and transformed into BL21 cells. After optimizing expression conditions, hnRNP A1 protein was purified using nickel column chromatography and identified by Western Blot. The purified protein was used to immunize C57BL/6 mice to produce polyclonal antibodies, and the antibody titer was determined by indirect ELISA.Results The highest expression of hnRNP A1 was achieved under conditions of 0.4 mM IPTG, induction temperature of 42°C, and induction time of 8 hours. The purified protein concentration reached 2.0563 μg/μl, and Western Blot confirmed the target protein. The antibody titer detected by indirect ELISA was 1:409,600.Conclusion The physicochemical properties of hnRNP A1 were successfully analyzed, high-efficiency expression of hnRNP A1 protein was achieved, and high-titer mouse-derived polyclonal antibodies against hnRNP A1 were prepared, providing a valuable tool for further research.
description: Objective This study aims to construct a recombinant expression plasmid using methods such as PCR and double enzyme digestion. By conducting single-factor experiments to adjust IPTG concentration, expression time, and expression temperature, the research seeks to optimize the expression conditions of the hnRNP A1 protein in BL21 competent cells. The goal is to obtain high-concentration, high-quality purified protein and to prepare high-titer polyclonal antibodies against hnRNP A1.Methods Bioinformatics tools were used to analyze the physicochemical properties and structure of hnRNP A1. The pET-28a-hnRNP A1 recombinant plasmid was constructed and transformed into BL21 cells. After optimizing expression conditions, hnRNP A1 protein was purified using nickel column chromatography and identified by Western Blot. The purified protein was used to immunize C57BL/6 mice to produce polyclonal antibodies, and the antibody titer was determined by indirect ELISA.Results The highest expression of hnRNP A1 was achieved under conditions of 0.4 mM IPTG, induction temperature of 42°C, and induction time of 8 hours. The purified protein concentration reached 2.0563 μg/μl, and Western Blot confirmed the target protein. The antibody titer detected by indirect ELISA was 1:409,600.Conclusion The physicochemical properties of hnRNP A1 were successfully analyzed, high-efficiency expression of hnRNP A1 protein was achieved, and high-titer mouse-derived polyclonal antibodies against hnRNP A1 were prepared, providing a valuable tool for further research.