Prokaryotic Expression and Polyclonal Antibody Preparation of Coxiella burnetii Omp34 Protein
WANG Nan1,2,3,5, WANG Xin1,2,3, WU Ding-Ding1,2,3, ZHAO Na1,2,3, DOU Zhi-Hao6, GAO Ming-Shan4, HAN Qin4, LIU Shi-Dong4, GAO Qing-Wu4, XUE Jun-Fei1,2,3, LI Zhao-Cai5, ZHAI Jun-Jun1,2,3,*, ZHANG Yan1,2,3,*
1 Shaanxi Province Engineering & Technology Research Center of Cashmere Goat, Yulin University, Yulin 719000, China; 2 College of Advanced Agricultural Sciences, Yulin University, Yulin 719000, China; 3 Yulin City Key Laboratory of Veterinary Public Health and Biosafety, Yulin 719000, China; 4 Hengshan District Animal Disease Prevention and Control Center, Yulin 719100, China; 5 State Key Laboratory for Animal Disease Control and Prevention/Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China; 6 College of Animal Science and Technology, Hebei Normal University of Science & Technology, Qinhuangdao 066004, China
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.
[1] 范德生, 徐琪毅, 禹惠兰, 等. 2011. 新疆伊犁地区人及家畜贝氏柯克斯体血清学调查[J]. 疾病监测, 26(1): 15-17. (Fan D S, Xu Q Y, Yu H L, et al.2011. Serological survey of Coxiella burnetii in humans and livestock in Yili Prefecture, Xinjiang[J]. Disease Surveillance, 26(1): 15-17.) [2] 韩翔舒, 宫震, 刘淑悦, 等. 2025. 牛冠状病毒S蛋白生物信息学分析及其受体结合域的多克隆抗体制备[J]. 畜牧与兽医, 57(8): 69-77. (Han X S, Gong Z, Liu S Y, et al.2025. Bioinformatics analysis of bovine Coronavirus S protein and preparation of polyclonal antibody against its receptor-binding domain[J]. Animal Husbandry and Veterinary Medicine, 57(8): 69-77.) [3] 李进春, 尹家祥. 2024. 贝氏柯克斯体特征及重要宿主啮齿动物感染现状[J]. 中国兽医学报, 44(2): 371-376. (Li J C, Yin J X.2024. Characteristics of Coxiella burnetii and infection status in rodents as important hosts[J]. Chinese Journal of Veterinary Science, 44(2): 371-376.) [4] 刘玲, 李璟, 范蕾, 等. 2022. 新冠病毒Nsp1蛋白结构与功能的生物信息学分析及原核表达[J]. 中国人兽共患病学报, 38(7): 566-576. (Liu L, Li J, Fan L, et al.2022. Bioinformatics analysis of structure and function of SARS-CoV-2 Nsp1 protein and its prokaryotic expression[J]. Chinese Journal of Zoonoses, 38(7): 566-576.) [5] 马玉惠, 陈惠琴, 李金新, 等. 2026. 细粒棘球蚴重组蛋白FGFR1的原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 53(7): 3811-3821. (Ma Y H, Chen H Q, Li J X, et al.2026. Prokaryotic expression and polyclonal antibody preparation of recombinant FGFR protein from Echinococcus granulosus[J]. Chinese Journal of Animal Husbandry and Veterinary Medicine, 53(7): 3811-3821.) [6] 乔海燕. 2013. 贝氏柯克斯体OmpH与ArgR外膜蛋白基因的原核表达及免疫原性分析[D]. 硕士学位论文, 山西农业大学, 导师: 张映, pp. 7-11. (Qiao H Y.2013. Prokaryotic expression and immunogenicity analysis of OmpH and ArgR outer membrane protein genes of Coxiella burnetii[D]. Thesis for M.S., Shanxi Agricultural University, Supervisor: Zhang Y, pp. 7-11.) [7] 孙长俭, 周连庆, 薛文成, 等. 2016. 辽宁省农村地区Q热立克次体血清学及虫媒蜱携带病原体状况调查[J]. 华南国防医学杂志, 30(6): 362-364. (Sun C J, Zhou L Q, Xue W C, et al.2016. Serological survey of Q fever rickettsia and investigation of pathogen carriage by vector ticks in rural areas of Liaoning Province[J]. Military Medical Journal of South China, 30(6): 362-364.) [8] 孙翔翔, 朱琳, 陈伟. 2019. Q热诊断技术研究进展[J]. 中国动物检疫, 36(5): 57-60. (Sun X X, Zhu L, Chen W.2019. Research progress on diagnostic technologies of Q fever[J]. China Animal Health Inspection, 36(5): 57-60.) [9] 谭启东, 张晓轩, 周东辉, 等. 2017. 宁夏回族自治区奶牛感染贝氏柯克斯体的血清流行病学调查[J]. 中国人兽共患病学报, 33(4): 349-352. (Tan Q D, Zhang X X, Zhou D H, et al.2017. Serological epidemiological survey of Coxiella burnetii infection in dairy cows in Ningxia Hui Autonomous Region[J]. Chinese Journal of Zoonoses, 33(4): 349-352.) [10] 武文君, 贾广乐, 廖娟红, 等. 2014. Q热贝氏柯克斯体间接ELISA检测方法的建立[J]. 中国畜牧兽医, 41(4):95-101. (Wu W J, Jia G L, Liao J H, et al.2014. Establishment of indirect ELISA for detection of Coxiella burnetii causing Q fever[J]. Chinese Journal of Animal Husbandry and Veterinary Medicine, 41(4): 95-101.) [11] 王锡乐, 邢洪光, 熊小路, 等. 2009. 氯仿-甲醇提取贝氏柯克斯体组分Q热疫苗的免疫保护性评价[J].中国人兽共患病学报, 25(3): 207-209+213. (Wang X L, Xing H G, Xiong X L, et al.2009. Evaluation of immune protection of Q fever vaccine prepared by chloroform-methanol extracted Coxiella burnetii components[J]. Chinese Journal of Zoonoses, 25(3): 207-209+213.) [12] 文英会, 崔鸿博, 陈曦艋, 等. 2025. 猪圆环病毒4型Cap蛋白的原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 52(10): 4904-4911. (Wen Y H, Cui H B, Chen X M, et al.2025. Prokaryotic expression of Cap protein of Porcine circovirus type 4 and preparation of polyclonal antibody[J]. Chinese Journal of Animal Husbandry and Veterinary Medicine, 52(10): 4904-4911.) [13] 熊小路, 焦俊, 温博海. 2018. Q热疫苗研究[J]. 中国人兽共患病学报, 34(2): 171-174+181. (Xiong X L, Jiao J, Wen B H.2018. Research progress on Q fever vaccines[J]. Chinese Journal of Zoonoses, 34(2): 171-174+181.) [14] 杨鸿玮, 靳换, 涂敏, 等. 2026. 鸽腺病毒1型Fiber-2蛋白的原核表达及多克隆抗体制备[J].中国畜牧兽医, 53(8): 4393-4402. (Yang H W, Jin H, Tu M, et al.2026. Prokaryotic expression and polyclonal antibody preparation of Fiber-2 protein from pigeon adenovirus type 1[J]. Chinese Journal of Animal Husbandry and Veterinary Medicine, 53(8): 4393-4402.) [15] 张卫斌, 吕长坤, 杨颖, 等. 2025. 人A组轮状病毒VP6蛋白的原核表达及其小鼠多克隆抗体的制备[J]. 中国人兽共患病学报, 41(09): 910-916. (Zhang W B, Lü C K, Yang Y, et al.2025. Prokaryotic expression of human rotavirus VP6 protein and preparation of mouse polyclonal antibodies[J]. Chinese Journal of Zoonoses, 41(09): 910-916.) [16] 张锐铮. 2024. 山羊源贝氏柯克斯体荧光定量PCR检测方法和抗体间接ELISA检测方法的建立及应用[D]. 硕士学位论文, 西北农林科技大学, 导师: 许信刚, pp. 1-4. (Zhang R Z.2024. Establishment and application of fluorescent quantitative PCR detection method and indirect ELISA detection method for Coxiella burnetii from goats[D]. Thesis for M.S., Northwest A&F University, Supervisor: Xu X G, pp. 1-4.) [17] Abeykoon A M H, Clark N J, Soares Magalhaes R J, et al.2021. Coxiella burnetii in the environment: A systematic review and critical appraisal of sampling methods[J]. Zoonoses Public Health, 68(3): 165-181. [18] Adhvaryu H, Huang L, Long C M, et al.2025. The intracellular agent of Q fever, Coxiella burnetii, alters human alveolar macrophage metabolism and mitochondrial physiology[J]. mBio, 16(9): e0143625. [19] Bauer B U, Schoneberg C, Herms T L, et al.2022. Impact of Coxiella burnetii vaccination on humoral immune response, vaginal shedding, and lamb mortality in naturally pre-infected sheep[J]. Frontiers in Veterinary Science, 9: 1064763. [20] Celina S S, Cerný J.2022. Coxiella burnetii in ticks, livestock, pets and wildlife: A mini-review[J]. Frontiers in Veterinary Science, 9: 1068129. [21] Cho Y S, Park J H, Kim J W, et al.2023. Current status of Q fever and the challenge of outbreak preparedness in Korea: One health approach to zoonoses[J]. Journal of Korean Medical Science, 38(24): e197. [22] Christodoulou M, Papagiannis D.2025. Q fever vaccines: Unveiling the historical journey and contemporary innovations in vaccine development[J]. Vaccines (Basel), 13(2): 151. [23] España P P, Uranga A, Cillóniz C, et al.2020. Q fever (Coxiella burnetii)[J]. Seminars in Respiratory and Critical Care Medicine, 41(4): 509-521. [24] Espí A, Del C A, Oleaga Á, et al.2021. One health approach: An overview of Q fever in livestock, wildlife and humans in Asturias (Northwestern Spain)[J]. Animals, 11(5): 1395. [25] Ferrara G, Colitti B, Pagnini U, et al.2022. Characterization of recombinant Ybgf protein for the detection of Coxiella antibodies in ruminants[J]. Journal of Veterinary Diagnostic Investigation, 34(4): 646-653. [26] Frangoulidis D, Kahlhofer C, Said A S, et al.2021. High prevalence and new genotype of Coxiella burnetii in ticks infesting camels in Somalia[J]. Pathogens, 10(6): 741. [27] Jahangiri A, Rasooli I, Owlia P, et al.2018. Highly conserved exposed immunogenic peptides of Omp34 against Acinetobacter baumannii: An innovative approach[J]. Journal of Microbiological Methods, 144: 79-85. [28] Jaltotage B, Ali U, Dorai-Raj A, et al.2021.Q fever endocarditis: A review of local and all reported cases in the literature[J]. Heart, Lung and Circulation, 30(10): 1509-1515. [29] Jemilehin F O, Okunlade A O, Adesola R O, et al.2026. Q fever in the 21st century: Uncovering diagnostic, epidemiological, and one health gaps in a re-emerging zoonosis[J]. Research in Veterinary Science, 201: 106060. [30] Larson C L, Pullman W, Beare P A, et al.2023. Identification of type 4B secretion system substrates that are conserved among Coxiella burnetii genomes and promote intracellular growth[J]. Microbiology Spectrum, 11(3): e0069623. [31] Mauermeir M, Ölke M, Hayek I, et al.2023. Bovine blood-derived macrophages are unable to control Coxiella burnetii replication under hypoxic conditions[J]. Frontiers in Immunology, 14: 960927. [32] Meles D K, Khairullah A R, Mustofa I, et al.2024. Navigating Q fever: Current perspectives and challenges in outbreak preparedness[J]. Open Veterinary Journal, 14(10): 2509-2524. [33] Miller H K, Priestley R A, Kersh G J.2020. Transmission of Coxiella burnetii by ingestion in mice[J]. Epidemiology and Infection, 148: e21. [34] Miller H K, Priestley R A, Kersh G J.2021. Q fever: A troubling disease and a challenging diagnosis[J]. Clinical Microbiology Newsletter, 43(13): 109-118. [35] Minder A, Kampouri E, Boillat-Blanco N, et al.2025. Q fever: What's new?[J]. Revue Médicale Suisse, 21(913): 730-735. [36] Mohammadi M R, Moradkasani S, Latifian M, et al.2025. Coxiella burnetii: Emerging threats, molecular insights, and advances in diagnosis and control measures[J]. Journal of Microbiological Methods, 237: 107213. [37] Sağlam A G, Çelik E, Büyük F, et al.2025. Coxiella burnetii in dogs and ticks from the Northeastern Anatolia region of Türkiye: Serological and molecular findings[J]. Veterinary Research Forum, 16(8): 431-437. [38] Sam G, Stenos J, Graves S R, et al.2023. Q fever immunology: The quest for a safe and effective vaccine[J]. NPJ Vaccines, 8(1): 133. [39] Shaw E I, Voth D E.2019. Coxiella burnetii: A pathogenic intracellular acidophile[J]. Microbiology, 165(1): 1-3. [40] Škultéty L.2020. Q fever and prevention[J]. Epidemiologie, Mikrobiologie, Imunologie, 69(2): 87-94. [41] Sobotta K, Bonkowski K, Liebler-Tenorio E, et al.2017. Permissiveness of bovine epithelial cells from lung, intestine, placenta and udder for infection with Coxiella burnetii[J]. Veterinary Research, 48(1): 23. [42] Steiner S, Roy C R.2024. CRISPR-Cas9-based approaches for genetic analysis and epistatic interaction studies in Coxiella burnetii[J]. mSphere, 9(12): e0052324. [43] Tan T, Heller J, Firestone S, et al.2023. A systematic review of global Q fever outbreaks[J]. One Health, 18: 100667. [44] Ullah Q, Jamil T, Saqib M, et al.2022. Q fever—a neglected zoonosis[J]. Microorganisms, 10(8): 1530. [45] Wan W, Zhang S, Zhao M, et al.2024. Lysosomal trafficking regulator restricts intracellular growth of Coxiella burnetii by inhibiting the expansion of Coxiella-containing vacuole and upregulating nos2 expression[J]. Frontiers in Cellular and Infection Microbiology, 13: 1336600.
