关键词: 羊源肺炎克雷伯菌, IutA蛋白, 原核表达, 生物信息学分析

Abstract: [Objective] To analyze the bioinformatics characteristics of the IutA protein from Klebsiella pneumoniae of ovine origin, providing a theoretical basis for its research as a candidate subunit vaccine protein. [Methods] Bioinformatics software was utilized to predict the physicochemical properties and structural characteristics of the IutA protein. Using the genomic DNA of K. pneumoniae as a template, the IutA gene was amplified via PCR. The recombinant expression plasmid pET-32a-IutA was constructed. Positive recombinant vectors were identified by double-enzyme digestion electrophoresis and gene sequencing. The verified plasmid was transformed into Escherichia coli BL21 （DE3） to induce recombinant protein expression. SDS-PAGE and Western blot were employed to detect protein expression. [Results] Bioinformatics prediction results indicated that the IutA protein consists of 731 amino acids, with a relative molecular mass of approximately 80.72 kDa and a theoretical isoelectric point of 4.80, classifying it as a stable hydrophilic protein. The protein contains one signal peptide and 22 transmembrane β-barrel domain. The amino acids at positions 44-731 belong to the conserved domain of the TONB-dependent siderophore receptor. The protein possesses 28 B-cell epitopes. Enzymatic digestion and sequencing confirmed the correct construction of the recombinant plasmid. SDS-PAGE and Western blot analyses revealed a specific target protein band of approximately 91 kDa, with the expression products predominantly present in inclusion bodies. [Conclusion] The physicochemical properties and structural characteristics of the IutA protein were obtained through bioinformatics prediction. The recombinant plasmid pET-32a-IutA was successfully constructed, and IutA protein expression was achieved in E. coli BL21 （DE3）, with the expression products existing mainly in the form of inclusion bodies. This study provides a foundation for subsequent evaluation of the immunogenicity of the IutA protein and the development of its subunit vaccine.

Key words: Klebsiella pneumoniae of ovine origin, IutA protein, prokaryotic expression, bioinformatics analysis