绵羊肺炎支原体EF-Tu蛋白的原核表达及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2024.02.025

Abstract

Abstract: 【Objective】 The purpose of this study was to clone EF-Tu gene in Mycoplasma ovipneumoniae (Mo), obtain EF-Tu protein by prokaryotic expression, prepare rabbit-derived polyclonal antibody against EF-Tu protein, laying the foundation for the study of the structure and function of Mo EF-Tu protein.【Method】 TGA codon in the middle of EF-Tu gene in pET-28a-EF-Tu plasmid was mutated to TGG by overlapping extension PCR, and the similarity alignment and genetic evolution analysis were conducted between the sequencing results and other Mycoplasma reference strains, and the putative protein sequences were analyzed for bioinformatics using online software.The mutated recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells for prokaryotic expression, identified by SDS-PAGE and Western blotting, and purified using Ni-chelating affinity chromatography.The polyclonal antibodies were prepared by immunizing rabbits with the purified EF-Tu fusion protein.The polyclonal antibody potency and immunological reactivity were detected by indirect ELISA and Western blotting.【Result】 The TGA site in EF-Tu gene was successfully mutated, and a prokaryotic expression vector expressing the His tag pET-28a-EF-Tu' was constructed.Bioinformatics analysis results showed that the cloned EF-Tu gene had the highest similarity and the closest genetic relationship to Mycoplasma ovipneumoniae strain MoGH3-3, and encoded 387 amino acids, without N-glycosylation site and transmembrane region.There were 10 serine, 20 threonine and 4 tyrosine phosphorylation sites of EF-Tu protein, and the secondary structure consists of random coil (35.14%), alpha helix (26.87%), extended chain (26.87%) and beta turn (11.11%).SDS-PAGE and Western blotting results showed that the size of the target protein was about 43 ku, and the protein purification concentration was 0.615 g/L.ELISA and Western blotting results showed that the prepared polyclonal antibody had a potency of up to 1∶128 000, and could specifically recognize the EF-Tu fusion protein, which had good immunological reactivity.【Conclusion】 The TGA codon of EF-Tu gene was successfully mutated in this study, the EF-Tu fusion protein was obtained by prokaryotic expression and purification, and its polyclonal antibody was prepared with a potency of 1∶128 000.This result provided an experimental basis for the subsequent study of the structure and biological function of Mo EF-Tu protein and its vaccine development.

Key words: Mycoplasma ovipneumoniae; overlapping extension PCR; EF-Tu gene; polyclonal antibody

CLC Number:

S852.62

WANG Yongfei, DENG Bowen, LIU Xiaoyan, HAERLEHA·Amantai, GUO Jiadong, ZHOU Zhengguo, CAI Jiang, LI Youwen. Prokaryotic Expression of EF-Tu Protein of Mycoplasma ovipneumoniae and Preparation of Polyclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(2): 689-699.

References

[1] 马玉,乔军,孟庆玲,等.绵羊肺炎支原体新疆流行株的分离鉴定及其膜蛋白P80基因序列分析[J].西南农业学报,2015,28(3):1375-1380. MA Y,QIAO J,MENG Q L,et al.Isolation and identification of Mycoplasma ovipneumoniae Xinjiang isolates and its sequence analysis of P80 gene[J].Southwest China Journal of Agricultural Sciences,2015,28(3):1375-1380.(in Chinese)
[2] 袁婷.绵羊肺炎支原体菌株的筛选及人工感染试验[D].石河子:石河子大学,2020. YUAN T.The screening and artificial infection of Mycoplasma ovipneumoniae[D].Shihezi:Shihezi University,2020.(in Chinese)
[3] MAKSIMOVIC Z,RIFATBEGOVIC M,LORIA G R,et al. Mycoplasma ovipneumoniae:A most variable pathogen[J].Pathogens,2022,11(12):1477.
[4] MACKAY J M K,NISBET D I,FOGGIE A.Isolation of pleuropneumonia-like organisms (genus Mycoplasma) from cases of sheep pulmonary adenomatosis (SPA)[J].Veterinary Record,1963,75(21):550-551.
[5] 胡景韶,蒋学良,胡诚隆,等.一种由支原体感染的绵羊增生性间质性肺炎的研究[J].中国兽医杂志,1982,5:2-6. HU J S,JIANG X L,HU C L.A study of proliferative interstitial pneumonia in sheep infected by Mycoplasma mycoides[J].Chinese Journal of Veterinary Medicine,1982,5:2-6.(in Chinese)
[6] 刘松艳,周薇,司微,等.肠炎沙门菌延伸因子EF-Tu缺失株对环境压力的应答变化[J].中国预防兽医学报,2016,38(1):31-34. LIU S Y,ZHOU W,SI W,et al.Biological characteristics and stress response of EF-Tu defective mutant of Salmonella enterica[J].Chinese Journal of Preventive Veterinary Medicine,2016,38(1):31-34.(in Chinese)
[7] 张洁,曹军军,祝明松,等.基于绵羊肺炎支原体EF-Tu蛋白间接ELISA方法的建立[J].动物医学进展,2019,40(11):6-13. ZHANG J,CAO J J,ZHU M S,et al.Establishment of an indirect ELISA method based on Mycoplasma ovipneumoniae elongation factor Tu protein[J].Progress in Veterinary Medicine,2019,40(11):6-13.(in Chinese)
[8] ZHANG Z X,HE M R,JIANG J M,et al.First report and comparative genomic analysis of Mycoplasma capricolum subsp. Capricolum HN-B in Hainan island,China[J].Microorganisms,2022,10(11):2298.
[9] ZHANG X,CHU Y F,ZHAO P,et al.Characterization of elongation factor Tu of Mycoplasma ovipneumoniae[J].Pakistan Veterinary Journal,2013,33(4):515-519.
[10] 焦凤娟,刘俊杰,卢思维,等.重叠延伸PCR技术定点突变TFEB原核表达载体的构建及体外诱导表达和纯化[J].吉林大学学报(医学版),2022,48(5):1109-1115. JIAO F J,LIU J J,LU S W,et al.Construction of prokaryotic expression vector of site directed mutant TFEB by SOE PCR technique and its induced expression and purification in vitro[J].Journal of Jilin University (Medicine Edition),2022,48(5):1109-1115.(in Chinese)
[11] 秦智,陈彦伯,李雨婷,等.重叠延伸PCR技术在单碱基定点突变中的作用[J].分子植物育种,2021,19(4):1157-1162. QIN Z,CHEN Y B,LI Y T,et al.Effect of overlapping extension PCR on single-base site-directed mutation[J].Molecular Plant Breeding,2021,19(4):1157-1162.(in Chinese)
[12] 周瑶琴,游凤,钟杰,等.一种快速鉴定猪肺炎支原体免疫显性蛋白抗原的ELISA方法的建立[J].生物工程学报,2018,34(1):44-53. ZHOU Y Q,YOU F,ZHONG J,et al.Development of an ELISA for identification of immunodominant protein antigens of Mycoplasma hyopneumoniae[J].Chinese Journal of Biotechnology,2018,34(1):44-53.(in Chinese)
[13] BARBOSA M S,MARQUES L M,TIMENETSKY J,et al.Host cell interactions of novel antigenic membrane proteins of Mycoplasma agalactiae[J].BMC Microbiology,2022,22(1):93.
[14] 戴伶俐,王娜,张帆,等.绵羊肺炎支原体分子致病机理研究进展[J].畜牧与饲料科学,2022,43(2):124-128. DAI L L,WANG N,ZHANG F,et al.Research progress in molecular pathogenic mechanism of Mycoplasma ovipneumoniae[J].Animal Husbandry and Feed Science,2022,43(2):124-128.(in Chinese)
[15] WIDJAJA M,HARVEY K L,HAGEMANN L,et al.Elongation factor Tu is a multifunctional and processed moonlighting protein[J].Scientific Reports,2017,7(1):11227.
[16] XIA X J,WANG L,CHENG L K,et al.Expression and immunological evaluation of elongation factor Tu of Streptococcus suis serotype 2[J].Polish Journal of Veterinary Sciences,2017,20(2):277-284.
[17] CHIU K H,WANG L H,TSAI T T,et al.Secretomic analysis of host-pathogen interactions reveals that elongation factor-Tu is a potential adherence factor of Helicobacter pylori during pathogenesis[J].Journal of Proteome Research,2017,16(1):264-273.
[18] HARVEY K L,JAROCKI V M,CHARLES I G,et al.The diverse functional roles of elongation factor Tu (EF-Tu) in microbial pathogenesis[J].Frontiers in Microbiology,2019,10:2351.
[19] JIANG F,HE J Y,NAVARRO-ALVAREZ N,et al.Elongation factor Tu and heat shock protein 70 are membrane-associated proteins from Mycoplasma ovipneumoniae capable of inducing strong immune response in mice[J].PLoS One,2016,11(8):e161170.
[20] QIAO M F,CHEN C,YANG S F,et al.Identification and characterization of immunogenic genes from genomic expression library of Mycoplasma ovipneumoniae[J].Kafkas Universitesi Veteriner Fakultesi Dergisi,2020,26(2):201-207.
[21] 刘威,袁芳艳,周丹娜,等.猪肺炎支原体脂蛋白LppT的多克隆抗体制备及鉴定[J].江苏农业科学,2017,45(19):220-223. LIU W,YUAN F Y,ZHOU D N,et al.Preparation and characterisation of a polyclonal antibody to the porcine Mycoplasma hyopneumoniae lipoprotein LppT[J].Jiangsu Agricultural Sciences,2017,45(19):220-223.(in Chinese)
[22] 周怡,王柏林,杨美,等.绵羊肺炎支原体pcDNA3.1-TBP30-Hsp70融合表达质粒的构建及对小鼠细胞免疫应答的影响[J].中国畜牧兽医,2019,46(11):3387-3395. ZHOU Y,WANG B L,YANG M,et al.Construction of Mycoplasma ovipneumoniae pcDNA3.1-TBP30-Hsp70 fusion plasmid and its effect on cellular immune response in mice[J].China Animal Husbandry&Veterinary Medicine,2019,46(11):3387-3395.(in Chinese)
[23] 尹正军.基于绵羊肺炎支原体tuf基因的荧光定量PCR方法的建立与应用[D].成都:西南民族大学,2015. YIN Z J.Development a Real-time PCR target tuf of Mycoplasma ovipneumoniae and application[D].Chengdu:Southwest Minzu University,2015.(in Chinese)
[24] 田睿.绵羊肺炎支原体IK3-3株免疫原性蛋白的筛选及鉴定[D].太谷:山西农业大学,2020. TIAN R.Screening and identification of immunogenicity proteins Mycoplasma ovipneumoniae strain IK3-3[D].Taigu:Shanxi Agricultural University,2020.(in Chinese)
[25] YU Y F,WANG J,HAN R,et al.Mycoplasma hyopneumoniae evades complement activation by binding to factor H via elongation factor thermo unstable (EF-Tu)[J].Virulence,2020,11(1):1059-1074.
[26] YU Y F,WANG H E,WANG J,et al.Elongation factor thermo unstable (EF-Tu) moonlights as an adhesin on the surface of Mycoplasma hyopneumoniae by binding to fibronectin[J].Frontiers in Microbiology,2018,9:974.

Prokaryotic Expression of EF-Tu Protein of Mycoplasma ovipneumoniae and Preparation of Polyclonal Antibody

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	REN Jianle, LIN Yiting, JI Kang, TAN Shanshan, CHEN Xinxin, JIN Yi, WANG Ying, NIU Sheng, LIANG Libin, LI Junping, ZHAO Yujun, TIAN Wenxia. Preparation of Polyclonal Antibody and Development of an Indirect ELISA Method for Senecavirus A VP2 Protein [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(2): 678-688.
[2]	REN Shengjie, SHEN Hongli, XIANG Xun, DAI Feiyan, ZHU Maoyin, WU Jiali, HU Zhihui, DUAN Gang, CHANG Hua. Polyclonal Antibody Preparation of TLR7 Protein and Its Expression Characteristics Analysis in DF-1 Cells of Larus ridibundus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2865-2875.
[3]	SUO Nanji, LI Peixuan, LIU Zhuanxia, WANG Tingwei, LIU Kejiang, XIE Dongxu, ZENG Weiwei, WANG Wenhui. Prokaryotic Expression and Polyclonal Antibody Preparation of Cyp-A in Bactrian Camel [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2896-2905.
[4]	LUO Shimei, ZHAO Yao, MA Xianping, ZHANG Jinyang, YI Huashan. Prokaryotic Expression and Polyclonal Antibody Preparation of Bluetongue Virus NS4 Protein [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2923-2930.
[5]	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.
[6]	OU Yunwen, PAN Qin, WANG Yang, DAI Junfei, REN Shaoke, ZHANG Yang, ZHANG Jie. Truncated Expression of ORF2 Gene of Porcine Parvovirus Type 6 and Preparation of Its Polyclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2487-2495.
[7]	WANG Xueping, ZHANG Xiaojun, LI Xiaoyue, WANG Guodong, ZHANG Fuliang, SONG Yuwei, ZHANG Mingliang, MA Lei. Prokaryotic Expression of Hexon Protein of Highly Pathogenic Fowl Adenovirus Serotype 4 and Preparation of Polyclonal Antibodies [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2053-2060.
[8]	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.
[9]	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.
[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]	WU Jun, HE Wenwen, PU Hao, JIN Min, SHI Wenyu, MA Aijun, LUO Tingxiang, YANG Depeng, Bayinchahan, HU Ercha. Expression, Bioinformatics Analysis of aqp3 Truncated Gene of Dermacentor marginatus and Preparation of Polyclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(11): 4589-4599.
[12]	CHEN Rujing, WU Xuemin, CHEN Qiuyong, YAN Shan, WU Renjie, CHE Yongliang, WANG Longbai, ZHOU Lunjiang. Prokaryotic Expression and Polyclonal Antibody Preparation of Porcine Circovirus Type 4 Rep Protein [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(10): 4188-4195.
[13]	ZHANG Mengyang, HE Jian, LIU Yangkun, YAO Lunguang. Bioinformatics Analysis and Polyclonal Antibody Preparation of African Swine Fever Virus p37 Protein [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 300-309.
[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]	XIAO Yangyang, MA Zhongchen, LI Ruirui, CHEN Chuangfu, ZHENG Wei, WANG Yong, WANG Pengyan. Bioinformatic Analysis and Polyclonal Antibody Preparation of Brucella Secreted Protein BspI [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 3112-3121.