贝氏柯克斯体Omp34蛋白原核表达及多克隆抗体制备

doi:10.3969/j.issn.1674-7968.2026.11.014

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Abstract Coxiella burnetii, the sole species of the genus Coxiella, is a microorganism with characteristics intermediate between viruses and bacteria. It can infect a wide range of hosts via multiple transmission routes and is the pathogen responsible for worldwide zoonotic Q fever (query fever). This study aimed to express recombinant outer membrane protein 34 (Omp34) from C. burnetii using a prokaryotic expression system, followed by purification to prepare rabbit polyclonal antibodies, which provided biological materials for establishing C. burnetii-specific serological detection methods and developing recombinant vaccines. The Omp34 gene was amplified from clinically positive samples using PCR. Bioinformatics analysis predicted the physicochemical properties and structural characteristics of the Omp34 protein. The recombinant plasmid pET-32a-Omp34 was constructed and transformed into Escherichia coli BL21 (DE3) competent cells. Following induction with isopropyl β-D-1-thiogalactopyranoside (IPTG), Omp34 protein was purified using Ni-IDA agarose column gravity purification and identified via SDS-PAGE and Western blot. Polyclonal antibodies were generated by immunizing healthy rabbits (Oryctolagus cuniculus) with Omp34 protein, and antibody titers were measured using indirect ELISA. A 918 bp fragment of the Omp34 gene from C. burnetii was obtained. Bioinformatics analysis revealed that this gene encoded 285 amino acids, with a protein molecular weight of approximately 32 kD, a theoretical isoelectric point of 5.67, an instability index of 48.96, an aliphatic index of 78.81, and a hydrophilicity of -0.869. No transmembrane domains or signal peptides were detected. The secondary structure of the Omp34 protein comprised α-helices (57.54%), extended chains (0.70%), and disordered coils (41.75%). Additionally, the protein contained 55 phosphorylation sites: 24 serine (Ser) phosphorylation sites, 23 threonine (Thr) phosphorylation sites, and 8 tyrosine (Tyr) phosphorylation sites. The molecular weight of the expressed recombinant protein matched the predicted 51.31 kD. After purification, the polyclonal antibody prepared from immunized rabbits (Oryctolagus cuniculus) reached a titer of 1∶12 800. This study provides a reference for exploring the biological functions of Omp34 and supplies critical materials for developing serological diagnostic methods for C. burnetii.

Key words： Coxiella burnetii Omp34 protein Prokaryotic expression Polyclonal antibody Bioinformatics analysis

Received: 10 March 2026

ZTFLH:

S852.64

Fund:陕西省科技厅中央引导地方科技发展资金项目(2025ZY-XCZXJX-12); 陕西省重点研发计划(2026HZ-YBXM-70); 国家自然科学基金地区科学基金(32060810; 31960710); 横山区2025年科技计划项目(YF2025-10); 榆阳区科技计划项目(2025-NY-15); 陕西省创新能力支撑计划项目(2021PT-017)

Corresponding Authors: *zhaijunjun1980@163.com; zhangyan1825@163.com

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	WANG Nan
	WANG Xin
	WU Ding-Ding
	ZHAO Na
	DOU Zhi-Hao
	GAO Ming-Shan
	HAN Qin
	LIU Shi-Dong
	GAO Qing-Wu
	XUE Jun-Fei
	LI Zhao-Cai
	ZHAI Jun-Jun
	ZHANG Yan

Cite this article:

WANG Nan,WANG Xin,WU Ding-Ding, et al. Prokaryotic Expression and Polyclonal Antibody Preparation of Coxiella burnetii Omp34 Protein[J]. 农业生物技术学报, 2026, 34(11): 2441-2453.

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https://journal05.magtech.org.cn/Jwk_ny/EN/10.3969/j.issn.1674-7968.2026.11.014 OR https://journal05.magtech.org.cn/Jwk_ny/EN/Y2026/V34/I11/2441

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