猪肺炎支原体P97蛋白单克隆抗体的制备、表位鉴定及其初步应用

doi:10.11843/j.issn.0366-6964.2020.05.022

Abstract

Abstract: In order to prepare a monoclonal antibody (MAb) against Mycoplasma hypneumoniae (Mhp), the splenic cells from BALB/c mice immunized with the P97 C-terminal region of Mhp J strain, which contains R1 region (P97CR1 protein) and was expressed in Escherichia coli (E. coli) BL21(DE3), were fused with SP2/0 myeloma cells, and a hybridoma cell line A3 stably secreting the MAbs against P97 Protein of Mhp were successfully screened. The identification results indicated the A3 MAb was IgG1 subclass with κ light chain. The A3 MAb was able to specifically recognize the P97CR1 protein of Mhp expressed in E. coli BL21(DE3) using Western blot analysis. Flow cytometry analysis showed that the MAb was able to recognize natural P97 protein expressed on the Mhp. The epitope sequence recognized by the A3 MAb was identified as LDDNLQby pepscan technique. The epitope sequence is highly conservative among the P97 proteins of all Mhp strains. The blocking ELISA analysis showed that the binding of the A3 MAb to the P97CR1 protein was blocked by high immune positive serum of Mhp. The preparation of A3 Mab provided foundation for further development of a blocking ELISA assay with high specificity and sensitivity for detecting Mhp infection.

Key words: Mycoplasma hypneumoniae (Mhp), monoclonal antibody (MAb), P97 protein, epitope identification

CLC Number:

WANG Ning, JIN Jin, LIU Lu, LIU Huanhuan, JIANG Libo, XIN Jiuqing, LIU Henggui. Preparation, Identification of the B-cell Epitope and Preliminary Application of a Monoclonal Antibody against P97 Protein of Mycoplasma hypneumoniae[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(5): 1110-1118.

References 28

[1]	MAES D, SIBILA M, KUHNERT P, et al. Update on Mycoplasma hyopneumoniae infections in pigs:knowledge gaps for improved disease control[J]. Transbound Emerg Dis, 2018, 65(S1):110-124.
[2]	武昱孜, 熊祺琰, 刘蓓蓓, 等. 猪肺炎支原体和猪鼻支原体双重荧光定量PCR检测方法的建立[J]. 畜牧兽医学报, 2017, 48(8):1491-1498.WU Y Z, XIONG Q Y, LIU B B, et al. Duplex real-time PCR method for detection of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis[J]. Acta Veterinaria et Zootechnica Sinica, 2017, 48(8):1491-1498. (in Chinese)
[3]	SIBILA M, NOFRARIAS M, LÓPEZ-SORIA S, et al. Chronological study of Mycoplasma hyopneumoniae infection, seroconversion and associated lung lesions in vaccinated and non-vaccinated pigs[J]. Vet Microbiol, 2007, 122(1-2):97-107.
[4]	MAES D, SEGALES J, MEYNS T, et al. Control of Mycoplasma hyopneumoniae infections in pigs[J]. Vet Microbiol, 2008, 126(4):297-309.
[5]	SIBILA M, PIETERS M, MOLITOR T, et al. Current perspectives on the diagnosis and epidemiology of Mycoplasma hyopneumoniae infection[J]. Vet J, 2009, 181(3):221-231.
[6]	THACKER E L. Diagnosis of Mycoplasma hyopneumoniae[J]. Anim Health Res Rev, 2004, 5(2):317-320.
[7]	WANG H Y, FENG Z X, WU Y Z, et al. The effects of Mycoplasma hyopneumoniae on porcine circovirus type 2 replication in vitro PK-15 cells[J]. Res Vet Sci, 2016, 105:56-61.
[8]	FENG Z X, SHAO G Q, LIU M J, et al. Immune responses to the attenuated Mycoplasma hyopneumoniae 168 strain vaccine by intrapulmonic immunization in piglets[J]. Agric Sci China, 2010, 9(3):423-431.
[9]	FISCH A, MARCHIORO S B, GOMES C K, et al. Commercial bacterins did not induce detectable levels of antibodies in mice against Mycoplasma hyopneumoniae antigens strongly recognized by swine immune system[J]. Trials Vaccinol, 2016, 5:32-37.
[10]	THACKER E L, THACKER B J, KUHN M, et al. Evaluation of local and systemic immune responses induced by intramuscular injection of a Mycoplasma hyopneumoniae bacterin to pigs[J]. Am J Vet Res, 2000, 61(11):1384-1389.
[11]	HAESEBROUCK F, PASMANS F, CHIERS K, et al. Efficacy of vaccines against bacterial diseases in swine:what can we expect?[J]. Vet Microbiol, 2004, 100(3-4):255-268.
[12]	MINION F C, ADAMS C, TSUNGDA H. R1 region of P97 mediates adherence of Mycoplasma hyopneumoniae to swine cilia[J]. Infect Immun, 2000, 68(5):3056-3060.
[13]	BARATE A K, CHO Y, TRUONG Q L, et al. Immunogenicity of IMS 1113 plus soluble subunit and chimeric proteins containing Mycoplasma hyopneumoniae P97 C-terminal repeat regions[J]. FEMS Microbiol Lett, 2014, 352(2):213-220.
[14]	SIMIONATTO S, MARCHIORO S B, MAES D, et al. Mycoplasma hyopneumoniae:from disease to vaccine development[J]. Vet Microbiol, 2013, 165(3-4):234-242.
[15]	MARCHIORO S B, FISCH A, GOMES C K, et al. Local and systemic immune responses induced by a recombinant chimeric protein containing Mycoplasma hyopneumoniae antigens fused to the B subunit of Escherichia coli heat-labile enterotoxin LTB[J]. Vet Microbiol, 2014, 173(1-2):166-171.
[16]	JENKINS C, WILTON J L, MINION F C, et al. Two domains within the Mycoplasma hyopneumoniae cilium adhesin bind heparin[J]. Infect Immun, 2006, 74(1):481-487.
[17]	HSU T, ARTIUSHIN S, MINION F C. Cloning and functional analysis of the P97 swine cilium adhesin gene of Mycoplasma hyopneumoniae[J]. J Bacteriol, 1997, 179(4):1317-1323.
[18]	CONCEICAO F R, MOREIRA  N, DELLAGOSTIN O A. A recombinant chimera composed of R1 repeat region of Mycoplasma hyopneumoniae P97 adhesin with Escherichia coli heat-labile enterotoxin B subunit elicits immune response in mice[J]. Vaccine, 2006, 24(29-30):5734-5743.
[19]	FENG Z X, SHAO G Q, LIU M J, et al. Development and validation of a SIgA-ELISA for the detection of Mycoplasma hyopneumoniae infection[J]. Vet Microbiol, 2010, 143(2-4):410-416.
[20]	FENG Z X, BAI Y, YAO J T, et al. Use of serological and mucosal immune responses to Mycoplasma hyopneumoniae antigens P97R1, P46 and P36 in the diagnosis of infection[J]. Vet J, 2014, 202(1):128-133.
[21]	沈青春, 王芳, 韩明远, 等. 猪肺炎支原体P46基因的原核表达与间接ELISA方法的建立[J]. 畜牧兽医学报, 2012, 43(3):431-437.SHEN Q C, WANG F, HAN M Y, et al. Expression of P46 protein of Mycoplasma hyopneumoniae in Escherichia coli and used for indirect ELISA[J]. Acta Veterinaria et Zootechnica Sinica, 2012, 43(3):431-437. (in Chinese)
[22]	肖增鸿, 黄昭亮, 林月霞, 等. 腹水型单克隆抗体纯化方法的研究[J]. 中国医药生物技术, 2013, 8(6):425-428.XIAO Z H, HUANG Z L, LIN Y X, et al. Study on purification methods of monoclonal antibody from mouse ascites fluid[J]. Chinese Medicinal Biotechnology, 2013, 8(6):425-428. (in Chinese)
[23]	贺佳慧, 白满元, 郭慧琛, 等. 兔抗O型口蹄疫VLPs酶标抗体的制备及初步应用[J]. 动物医学进展, 2019, 40(1):20-24.HE J H, BAI M Y, GUO H C, et al. Preparation of rabbit anti-type O foot-and-mouth disease virus VLPs IgG-HRP[J]. Progress in Veterinary Medicine, 2019, 40(1):20-24. (in Chinese)
[24]	张悦. 猪肺炎支原体P65蛋白的克隆表达、单克隆抗体制备及阻断ELISA抗体检测方法建立[D]. 南京:南京农业大学, 2013.ZHANG Y. Cloning and expression of P65 gene from Mycoplasma hyopneumoniae、preparation of the monoclonal antibody against P65 protein and establishment of blocking ELISA for antibody to Mycoplasma hyopneumoniae[D]. Nanjing:Nanjing Agricultural University, 2013. (in Chinese)
[25]	OKAMBA F R, ARELLA M, MUSIC N, et al. Potential use of a recombinant replication-defective adenovirus vector carrying the C-terminal portion of the P97 adhesin protein as a vaccine against Mycoplasma hyopneumoniae in swine[J]. Vaccine, 2010, 28(30):4802-4809.
[26]	LEE S H, LEE S, CHAE C, et al. A recombinant chimera comprising the R1 and R2 repeat regions of M. hyopneumoniae P97 and the N-terminal region of A. pleuropneumoniae ApxIII elicits immune responses[J]. BMC Vet Res, 2014, 10(1):43.
[27]	SHIMOJI Y, OISHI E, MUNETA Y, et al. Vaccine efficacy of the attenuated Erysipelothrix rhusiopathiae YS-19 expressing a recombinant protein of Mycoplasma hyopneumoniae P97 adhesin against mycoplasmal pneumonia of swine[J]. Vaccine, 2003, 21(5-6):532-537.
[28]	OGAWA Y, OISHI E, MUNETA Y, et al. Oral vaccination against mycoplasmal pneumonia of swine using a live Erysipelothrix rhusiopathiae vaccine strain as a vector[J]. Vaccine, 2009, 27(33):4543-4550.

Preparation, Identification of the B-cell Epitope and Preliminary Application of a Monoclonal Antibody against P97 Protein of Mycoplasma hypneumoniae

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