空肠弯曲菌PEB1A蛋白的原核表达及间接ELISA方法的建立

doi:10.16431/j.cnki.1671-7236.2017.12.030

Abstract

Abstract:

In order to develop an indirect ELISA method to detect Campylobacter jejuni antibody, PEB1A gene of Campylobacter jejuni was cloned and amplified by PCR, the prokaryotic expression vector pET32a-PEBIA was constructed, and then transferred into the expression strain E.coli BL21 (DE3), and obtained about 47 ku of soluble protein. Western blotting result showed that the expressed recombinant protein had good biological activity, an indirect ELISA method for detecting antibody against Campylobacter jejuni was developed using expressed PEB1A protein as coating antigen,and detected its specificity, sensitivity, repeatability, respectively. The results showed that the established method for detection of Campylobacter jejuni antibody critical value was 0.3424. This method only specifically reacts with Campylobacter jejuni positive sera, and had no cross-reactivity with other antiserum and strong specificity. In addition, the coefficients of variations in both inter-and intra-assay were less than 5% indicating that it had good repeatability and stability. The establishment of this method could be applied to the rapid detection of Campylobacter jejuni in serum, and provided basis for further prevention and control of Campylobacter jejuni diarrhea.

Key words: Campylobacter jejuni; indirect ELISA; adhesion protein PEB1A; prokaryotic expression

CLC Number:

S852.61⁺3

WANG Li, SHA Zhou, LI Shi-yu, LIANG Jia-ming, WANG Xing-long. Prokaryotic Expression of PEB1A Protein of Campylobacter jejuni and Establishment of an Indirect ELISA[J]. , 2017, 44(12): 3605-3611.

References

[1] Hadden R D M, Gregson N A.Guillain-Barré syndrome and Campylobacter jejuni infection[J]. Journal Applied Microbiology,2001, 90(56):145S-154S.
[2] Buzby J C, Roberts T, Ban M A. Estimated annual costs of campylobacter-associated Guillain-Barre syndrome[R]. Agricultural Economics Reports, 1997.
[3] 臧星星,马宝骊,曹鹤年,等.空肠弯曲菌蛋白抗原分析[J].中国人兽共患病学报,1995,11(2):2-6.
[4] Yao R, Burr D H, Guerry P. CheY-mediated modulation of Campylobacter jejuni virulence[J].Molecular Microbiology, 1997, 23(5):1021-1031.
[5] Leon-Kempis M R, Guccione E, Mulholland F, et al. The Campylobacter jejuni, PEB1a adhesin is an aspartate/glutamate-binding protein of an ABC transporter essential for microaerobic growth on dicarboxylic amino acids[J]. Molecular Microbiology, 2006, 60(5):1262-1275.
[6] 王婷.空肠弯曲菌flaA、PEB1A基因的克隆表达及单克隆抗体的制备[D].南京:南京农业大学, 2008.
[7] Liu G, Han Y, Li X, et al. Applicability of a rapid method based on immunomagnetic capture-fluorescent PCR assay for Campylobacter jejuni[J]. Food Control, 2006, 17(7):527-532.
[8] Sails A D, Fox A J, Bolton F J, et al. A Real-time PCR assay for the detection of Campylobacter jejuni in foods after enrichment culture[J]. Applied & Environmental Microbiology, 2003, 69(3):1383-1390.
[9] Sails A D, Fox A J, Bolton F J, et al. Development of a PCR ELISA assay for the identification ofCampylobacter jejuni, and Campylobacter coli[J]. Molecular & Cellular Probes, 2001, 15(5):291-300.
[10] Whiteduck-Léveillée J, Cloutier M, Topp E, et al. Development and evaluation of multiplex PCR assays for rapid detection of virulence-associated genes in Arcobacter species[J]. Journal of Microbiological Methods, 2016, 121:59-65.
[11] 肖斌,朱永红,邹全明.简便敏感的环介导等温扩增基因诊断新技术[J]. 中华检验医学杂志, 2005, 28(7):761-763.
[12] 刘丽娜, 邵华斌, 程国富,等. J亚群禽白血病病毒gp85基因的原核表达及间接ELISA方法建立[J].中国预防兽医学报, 2014, 36(1):38-41.
[13] Tsugawa T, Nikaido K, Doi T, et al. Guillain-Barre syndrome with meningoencephalitis after Campylobacter jejuni infection[J]. Pediatric Infectious Disease Journal, 2004, 23(10):966.
[14] Humphrey T J.Campylobacter jejuni:Some aspects of epidemiology, detection and control[J]. British Food Journal, 2013, 94(1):21-25.
[15] Jørgensen F, Bailey R, Williams S, et al. Prevalence and numbers of Salmonella and Campylobacter spp. on raw, whole chickens in relation to sampling methods[J]. International Journal of Food Microbiology, 2002, 76(1-2):151-164.
[16] 楼宏强, 胡野, 王岚,等. 空肠弯曲菌主要外膜蛋白OMP18的B细胞抗原表位分析及功能鉴定[J]. 中国人兽共患病学报, 2014, 30(7):739-742.
[17] 尹衍新. 基于CjaA蛋白检测鸡空肠弯曲菌抗体的间接ELISA方法建立及其单克隆抗体研制[D].扬州:扬州大学, 2010.
[18] Pei Z H, Rd E R, Blaser M J. Identification, purification, and characterization of major antigenic proteins of Campylobacter jejuni[J]. Journal of Biological Chemistry, 1991, 266(25):16363-16369.
[19] Kervella M, Pagès J M, Pei Z, et al. Isolation and characterization of two Campylobacter glycine-extracted proteins that bind to HeLa cell membranes[J]. Infection & Immunity, 1993, 61(8):3440-3448.
[20] 王丽珍, 罗桂芳, 张艳萍,等. 猪流行性腹泻病毒间接ELISA检测方法的建立[J]. 中国畜牧兽医, 2017,44(2):377-383.

Prokaryotic Expression of PEB1A Protein of Campylobacter jejuni and Establishment of an Indirect ELISA

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	JIAO Linlin, CHENG Yuting, WU Qingguo, WU Shuang, WU Zhi, ZHU Shanyuan, QIAN Yingjuan. Prokaryotic Expression and Polyclonal Antibody Preparation of Duck Tembusu Virus Capsid Protein [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2395-2402.
[2]	LI Can, LI Chuchu, ZENG Wei, ZHANG Ning, JI Chunxiao, CHEN Tao. Recombinant Expression and Function Study of RegⅢγ Protein in Porcine [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2414-2426.
[3]	SONG Yue, SU Shengjie, ZHANG Fan, BAI Fan, DAI Lingli, WANG Na, WANG Dawei, YANG Qianwen, ZHAO Shihua, ZHANG Yuemei. Prokaryotic Expression and Polyclonal Antibody Preparation of Mannheimia haemolytica Truncated Leukotoxin [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2479-2486.
[4]	ZHANG Fangyuan, LIN Yating, YANG Dawei, CHEN Hu, LI Guimei, SHAN Hu. Prokaryotic Expression of African Swine Fever Virus P30 Protein and Establishment of Indirect ELISA Detection Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2012-2022.
[5]	HU Leyu, HU Xinyao, LI Ying, ZHAO Shengmiao, SHEN Fengzhen, WANG Changfa, LI Liangliang, WANG Tongtong, REN Huiying. Bioinformatics Analysis of HO-1 Gene and Preparation of Polyclonal Antibody in Dezhou Donkey [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1499-1510.
[6]	WANG Wenjie, RU Penghui, YU Chuan, DU Fuxi, CHEN Songbiao, SHANG Ke, ZHANG Chunjie, CHENG Xiangchao. Prokaryotic Expression of VP2 Protein of Luoyang Feline Panleukopenia Virus Isolate and Polyclonal Antibody Preparation [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1511-1521.
[7]	LIU Jiaxing, HAN Xueying, ZHANG Xinru, DENG Jiaxin, YAO Lunguang, LIU Yangkun. Preparation and Immunogenicity Analysis of Ferritin Nanoparticles Carrying Antigenic Epitopes of Porcine Epidemic Diarrhea Virus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1567-1574.
[8]	JIA Xinyue, MA Jing, ZHANG Yanyan, MA Xun, WANG Zhengrong, BO Xinwen. Bioinformatics Analysis and Prokaryotic Expression of EGR-07243 and EGR-07243 Genes of Echinococcus granulosus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 847-858.
[9]	ZHANG Zhitao, LI Xianglong, KONG Lingli, LUO Yuxin, WANG Dongying. Prokaryotic Expression of Serine/Threonine Protein Phosphatase 2A in Fasciola gigantica and Expression Analysis at Different Stages [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1107-1117.
[10]	NIU Xiaojie, XU Mingguo, XIAO Li, LIU Wenhao, CHEN Chuangfu, WANG Yong. Expression and Purification of IPAJ Protein of Salmonella Pullorum, Preparation of Polyclonal Antibody and Establishment of ELISA Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1218-1228.
[11]	GAN Lu, ZHENG Huizhen, NUO Mingdalai, LIU Yan, JIN Min, HE Wenwen, WEN Licui, LI Yongchang, GAILIKE·Bayinchahan. Propagation of Trypanosoma evansi Yili Strain and Cloning, Expression and Bioinformatics Analysis of Its PFR Gene [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 469-478.
[12]	LYU Shuangfei, MA Xun, WANG Jing, KONG Cuilian, KOU Lijun, LIU Caixia, SHI Weidi, KANG Lichao, REN Huijie. Prokaryotic Expression and Immunoreactivity of PA-LF1 Fusion Protein of Bacillus anthracis [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 674-683.
[13]	ZHANG Kai, ZHANG Chunping, GE Shengqiang, SUN Chunxi, CHENG Jie, FU Chunyu, WANG Gang, NIU Xing, PENG Jun. Expression of p22 Protein of African Swine Fever Virus,Preparation of Polyclonal Antibody and Establishment of Indirect ELISA Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 270-279.
[14]	ZHU Lilin, WANG Houkun, CHEN Xueyang, LIU Jing, LIANG Xiongyan, FANG Chun, YANG Yuying. Effect of Chicken IFIT5 on Replication of J Subgroup Avian Leukemia Virus Within DF-1 Cells [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 310-316.
[15]	HAN Rui, YANG Xing, ZHANG Wenbo, CHEN Jialing, ZHOU Xiaoli. Prokaryotic Expression of Autolysin from Streptococcus suis and Establishment of an Indirect ELISA Method for Detection of Its Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(9): 3508-3519.