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1 44 生物工程学报 Chinese Journal of Biotechnology DOI: /j.cjb 动物及兽医生物技术周瑶琴等 / 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 Jan. 25, 2018, 34(1): Chin J Biotech, All rights reserved ELISA 周瑶琴, 游凤, 钟杰, 王豪举, 丁红雷 ,,,. ELISA., 2018, 34(1): Zhou YQ, You F, Zhong J, et al. Development of an ELISA for identification of immunodominant protein antigens of Mycoplasma hyopneumoniae. Chin J Biotech, 2018, 34(1): 摘要 : 旨在建立一种快速鉴定猪肺炎支原体 (Mycoplasma hyopneumoniae,mhp) 血清体液免疫显性蛋白抗原的方法通过构建 pgex-6p-1-mhp366 重组表达质粒并转入大肠杆菌 BL21(DE3), 将 GST-Mhp366 重组蛋白进行原核表达将 GST-Mhp366 重组菌和 GST 工程菌裂解液加入谷胱甘肽包被板进行抗原包被, 分别与 17 份 Mhp 阳性血清和 13 份 Mhp 阴性血清反应, 通过对抗原包被量封闭液血清和二抗稀释度的优化, 确定间接 ELISA 的反应条件最终确定 GST-Mhp366 重组菌和 GST 工程菌裂解液原液为抗原的最佳包被量,PBS+10% FBS+2.5% 脱脂奶粉为最佳封闭液, 分别将血清按照稀释酶标二抗按照稀释作为最佳工作浓度, 从而建立了一种基于间接 ELISA 的快速鉴定 Mhp 血清体液免疫显性蛋白抗原的方法, 同时通过已知的血清体液免疫显性蛋白 Mhp156 和 Mhp364 对所建立的方法进行了验证该方法的建立能够用于在基因组水平高通量筛选 Mhp 血清体液免疫显性蛋白抗原, 并为 Mhp 初乳体液免疫和黏膜免疫显性蛋白抗原鉴定方法的建立奠定基础 : 猪支原体肺炎,Mhp366, 免疫显性蛋白抗原, 酶联免疫吸附试验 Received: May 31, 2017; Accepted: November 16, 2017 Supported by: Fundamental Research Funds for the Central Universities (No. XDJK2017D034), Chongqing Special Project of Social Livelihood Science and Technology Innovation (No. cstc2015shmszx 80033). Corresponding author: Honglei Ding. Tel: ; hongleiding@swu.edu.cn (No. XDJK2017D034) (No. cstc2015shmszx80033)

2 周瑶琴等 / 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 45 Development of an ELISA for identification of immunodominant protein antigens of Mycoplasma hyopneumoniae Yaoqin Zhou, Feng You, Jie Zhong, Haoju Wang, and Honglei Ding Laboratory of Veterinary Lemology, Southwest University, Chongqing , China Abstract: We developed a method to identify serological humoral immunodominant proteinic antigen of Mycoplasma hyopneumoniae (Mhp). After constructing the recombinant plasmid pgex-6p-1-mhp366 and transforming it into Escherichia coli BL21(DE3), the recombinant GST-Mhp366 protein was expressed successfully. The lysates of the recombinant GST-Mhp366 and genetic engineering GST were added into glutathione coated plates and reacted with 17 positive sera or 13 negative sera. Meanwhile, the optimization of experimental conditions, including coated antigen, blocking buffer, dilutions of sera and second antibody were determined. The optimal concentration of the coated antigen was the original bacteria lysates without dilution, and the optimal blocking buffer contained 10% FBS and 2.5% skim milk in PBS. Besides, the working concentration of serum samples and the HRP-tagged rabbit anti-pig IgG secondary antibody were 1:500 and 1:40 000, respectively. Thus, an indirect ELISA was established for identification of immunodominant protein antigens of Mhp. Meanwhile, this method was confirmed by the identified serological humoral immunodominant proteinic antigen Mhp156 and Mhp364. This method can be used for identification of the candidate vaccine antigens on a genome-wide scale. Furthermore, it can lay the foundation for identifying the candidate vaccine antigens through colostra and the nasal mucosal secretions. Keywords: mycoplasmal pneumonia of swine, Mhp366, immunodominant antigen, enzyme-linked immunosorbent assay (Mycoplasmal pneumonia of swine MPS) (Mycoplasma hyopneumoniae Mhp) [1] 100 [2] MPS MPS [3-4] [4] [5-6] MPS [7] Mhp MPS Mhp366 Mhp [8] Mhp366 (Indirect enzyme-linked immunosorbent assay Indirect ELISA) Mhp Mhp156 Mhp364 [9] Mhp Mhp cjb@im.ac.cn

3 46 ISSN CN /Q Chin J Biotech 1 材料与方法 1.1 菌株和质粒 BL21(DE3) XL-1 Blue pgex-6p-1 mhp366 mhp364 mhp156 ( ) mhp mhp mhp TGA TGG pgex-6p-1 pgex-6p-1-mhp366 pgex-6p-1-mhp364 pgex- 6P-1-mhp156 DH5α 1.2 血清 Mhp 35 MPS Mhp 30 MPS Mhp 1.3 主要试剂和菌株 marker ( ) Omega BamHⅠ XhoⅠ SalⅠ marker ( ) GE Healthcare HRP IgG (FBS) Thermo Scientific ELISA IDEXX ELISA A B GST-Tag Bioworld HRP IgG Proteintech PVDF Roche Diagnostics eecl PreScission Protease 1.4 重组蛋白的原核表达与纯化 DH5α pgex-6p-1-mhp366 pgex-6p-1-mhp364 pgex-6p-1-mhp156 pgex-6p-1-mhp366 pgex-6p-1-mhp364 BamHⅠ XhoⅠ pgex-6p-1-mhp156 BamHⅠ SalⅠ pgex-6p-1-mhp366 pgex-6p-1-mhp364 BL21(DE3) pgex-6p-1-mhp156 XL-1 Blue BamHⅠ+XhoⅠ BamHⅠ+XhoⅠ BamHⅠ+SalⅠ GST-Mhp366 GST-Mhp364 GST-Mhp156 GST-Mhp366 GST-Mhp364 GST-Mhp μg/ml LB 37 OD IPTG 1 mmol/l GST-Mhp366 GST-Mhp h GST-Mhp h r/min 15 min (PBS 1% Triton X mmol/l 75 IU/mL 20 μmol/l 1.6 μmol/l ph 7.2) (220 V 110 W) 30 ( 3 s 4 s) r/min 15 min (25 r/min) 1 h PBST 2 PBS 1 SDS-PAGE PBS PreScission Protease 5 h r/min 1 min PBS

4 周瑶琴等 / 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 47 2 SDS-PAGE pgex-6p-1 BL21(DE3) GST IPTG GST-Mhp 重组蛋白的鉴定 GST-Mhp366 GST-Mhp364 GST-Mhp156 Western blotting SDS-PAGE PVDF 5% TBST 2 h TBST 3 5 min GST-Tag ( ) 1 h TBST 3 5 min HRP IgG ( ) 1 h TBST 3 5 min eecl 1.6 Mhp 阳性血清和阴性血清的准备 Mhp Mhp PCR [10] Mhp 17 Mhp Mhp 13 Mhp GST IPTG GST =1 50 Mhp / 4 (25 r/min) 1.7 最佳抗原包被浓度的选择 GST-Mhp ml 1 ml r/min 15 min μl 4 PBST 5 3 min 200 μl 0.25% 10% FBS PBS 1 h PBST 5 3 min μl 2 h PBST 5 3 min HRP IgG μl 37 1 h PBST 5 3 min A B 50 μl 10 min 50 μl 10 min OD 630 GST GST-Mhp366 ELISA OD 最佳封闭液的确定 GST-Mhp366 GST PBS+10% FBS PBS+2.5% PBS+10% FBS+2.5% 200 μl 1 h ELISA OD 最佳血清稀释度和最佳酶标二抗工作浓度的确定 GST-Mhp366 GST HRP IgG OD 间接 ELISA 反应条件的判定标准 ELISA GST-Mhp366 GST 13 Mhp GST-Mhp366 GST OD 630 x s x s Mhp366 GST-Mhp366 GST 17 Mhp GST-Mhp366 GST OD cjb@im.ac.cn

48 ISSN 1000-3061 CN 11-1998/Q Chin J Biotech A A x A x s Mhp366 s Mhp ELISA 1.11 间接 ELISA 方法的验证 GST-Mhp364 GST-Mhp156 25 ml 1 ml GST-Mhp366 ELISA [9] Mhp364 Mhp156 Mhp 2 结果与分析 2.

pgex-6p-1-mhp156 BL21(DE3)/XL-1 Blue GST 5 000 bp pgex-6p-1 BL21(DE3) GST-Mhp366 GST-Mhp364 GST-Mhp156 1 mmol/l IPTG 13 000 r/min 12 000 g [11] 3 GST-Mhp366 SDS-PAGE 90 kda 26 kda ( 2 1 ) 90 kda

5 48 ISSN CN /Q Chin J Biotech A A x A x s Mhp366 s Mhp ELISA 1.11 间接 ELISA 方法的验证 GST-Mhp364 GST-Mhp ml 1 ml GST-Mhp366 ELISA [9] Mhp364 Mhp156 Mhp 2 结果与分析 2.1 GST-Mhp366 GST-Mhp364 GST-Mhp156 和 GST 蛋白的原核表达纯化与鉴定 GST-Mhp366 GST-Mhp364 GST- Mhp bp bp bp bp mhp366 mhp364 mhp156 ( 1) pgex-6p-1-mhp366 pgex-6p-1-mhp364 pgex-6p-1-mhp156 BL21(DE3)/XL-1 Blue GST bp pgex-6p-1 BL21(DE3) GST-Mhp366 GST-Mhp364 GST-Mhp156 1 mmol/l IPTG r/min g [11] 3 GST-Mhp366 SDS-PAGE 90 kda 26 kda ( 2 1 ) 90 kda GST-Mhp kda GST PreScission Protease 70 kda Mhp366 ( ) (64 kda) - Mhp366 GST 46 kda PreScission Protease ( 2 6 ) GST-Mhp kda GST-Mhp kda GST ( 2 7 ) PreScission Protease 61 kda Mhp364 ( ) - Mhp364 GST 46 kda PreScission Protease ( 2 12 ) 图 1 pgex-6p-1-mhp366 pgex-6p-1-mhp364 pgex-6p-1-mhp156 重组质粒的双酶切鉴定 Fig. 1 Identification of recombinant plasmids pgex- 6P-1-mhp366, pgex-6p-1-mhp364 and pgex-6p-1-mhp156 by double enzyme digestion. M: DNA marker Ⅲ; Lane 1: pgex-6p-1-mhp366 was digested with BamHⅠ and XhoⅠ. Lane 2: pgex-6p-1-mhp364 was digested with BamHⅠ and XhoⅠ. Lane 3: pgex-6p-1-mhp156 was digested with BamHⅠ and SalⅠ.

M: protein molecular weight marker; 1: GST-Mhp366 fusion protein; 2: the Mhp366 supernatant was harvested after the cleavage; 3 5: the Mhp366 beads were sequentially washed three times with PBS; 6:

6 周瑶琴等 / 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 49 图 2 GST-Mhp366 GST-Mhp364 GST-Mhp156 重组蛋白表达纯化及蛋白酶切分析的电泳图 Fig. 2 Recombinant proteins GST-Mhp366, GST-Mhp364 and GST-Mhp156 were purified by Glutathione Sepharose TM 4B and digested by PreScission Protease. M: protein molecular weight marker; 1: GST-Mhp366 fusion protein; 2: the Mhp366 supernatant was harvested after the cleavage; 3 5: the Mhp366 beads were sequentially washed three times with PBS; 6: the remaining Mhp366 bead sample after digestion and washing; 7: GST-Mhp364 fusion protein; 8: the Mhp364 supernatant was harvested after the cleavage; 9 11: the Mhp364 beads were sequentially washed three times with PBS; 12: the remaining Mhp364 bead sample after digestion and washing; 13: GST-Mhp156 fusion protein; 14: the Mhp156 supernatant was harvested after the cleavage; 15 17: the Mhp156 beads were sequentially washed three times with PBS; 18: the remaining Mhp156 bead sample after digestion and washing. GST-Mhp kda GST-Mhp kda GST ( 2 13 ) PreScission Protease 36 kda Mhp156 ( ) - Mhp156 GST 46 kda PreScission Protease ( 2 18 ) GST Western blotting GST-Mhp366 GST-Mhp364 GST-Mhp kda ( 3) 间接 ELISA 反应条件的确定 GST-Mhp366 GST A x s Mhp366 ( 4) Mhp 图 3 GST-Mhp366 GST-Mhp364 GST-Mhp156 重组蛋白进行 Western blotting 分析 Fig. 3 Western blotting analysis of recombinant proteins GST-Mhp366, GST-Mhp364 and GST-Mhp156. M: precision Plus Protein Dual Color Standards; 1: GST- Mhp366 fusion protein; 2: GST-Mhp364 fusion protein; 3: GST-Mhp156 fusion protein cjb@im.ac.cn

50 ISSN 1000-3061 CN 11-1998/Q Chin J Biotech 13

(Mhp364 Mhp156 7 448 MHP0353 MHP0223) [9] 图 4

4 Optimization for the concentration of coating

5% ELISA A x s Mhp366 ( 5) PBS+10% FBS+2.

7 50 ISSN CN /Q Chin J Biotech 13 Mhp364 9 Mhp Mhp Mhp364 Mhp156 Simionatto (Mhp364 Mhp MHP0353 MHP0223) [9] 图 4 抗原最佳包被浓度的选择 Fig. 4 Optimization for the concentration of coating antigen with indirect ELISA. GST-Mhp PBS+10% FBS PBS+2.5% PBS+10% FBS+2.5% 3 PBS+10% FBS+2.5% ELISA A x s Mhp366 ( 5) PBS+10% FBS+2.5% Mhp ( 6) ELISA GST-Mhp364 GST- Mhp Mhp 图 5 最佳封闭液的确定 Fig. 5 Optimization of blocking buffer. 图 6 最佳血清稀释度和最佳酶标二抗工作浓度的确定 Fig. 6 Determination of optimal dilution of serum and HRP-conjugated IgG.

8 周瑶琴等 / 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 51 3 讨论 Mhp366 Mhp [8] Mhp366 TGG TGA [12] TGA mhp366 mhp364 mhp TGA Mhp366 Mhp364 Mhp156 TGA TGG PCR mhp366 mhp364 mhp156 TGA TGG 3 TGA TGG pgex-6p-1 mhp366 mhp364 mhp156 pgex-6p-1 GST GST Mhp366 Mhp364 Mhp156 [12] GST [13] ELISA GST ELISA ELISA GST GST GST [14] Mhp [15-17] [9,18] GST [19-21] [22] [23-24] Mhp GST-Mhp366 Mhp366 Mhp (16/17) Meens [8] Mhp Mhp364 Mhp156 [25] Mhp [9] Mhp364 Mhp156 Mhp Mhp cjb@im.ac.cn

9 52 ISSN CN /Q Chin J Biotech Mhp REFERENCES [1] Maes D, Segales J, Meyns T, et al. Control of Mycoplasma hyopneumoniae infections in pigs. Vet Microbiol, 2008, 126(4): [2] Chen C, Hua LZ, Gan Y, et al. Research progress of MPS eradication programme. China Animal Health, 2014, 16(11): (in Chinese).,,,.., 2014, 16(11): [3] Sibila M, Bernal R, Torrents D, et al. Effect of sow vaccination against Mycoplasma hyopneumoniae on sow and piglet colonization and seroconversion, and pig lung lesions at slaughter. Vet Microbiol, 2008, 127(1/2): [4] Meyns T, Dewulf J, de Kruif A, et al. Comparison of transmission of Mycoplasma hyopneumoniae in vaccinated and non-vaccinated populations. Vaccine, 2006, 24(49/50): [5] Villarreal I, Meyns T, Dewulf J, et al. The effect of vaccination on the transmission of Mycoplasma hyopneumoniae in pigs under field conditions. Vet J, 2011, 188(1): [6] Villarreal I, Maes D, Vranckx K, et al. Effect of vaccination of pigs against experimental infection with high and low virulence Mycoplasma hyopneumoniae strains. Vaccine, 2011, 29(9): [7] Singh K, Mehta S. The clinical development process for a novel preventive vaccine: an overview. J Postgrad Med, 2016, 62(1): [8] Meens J, Bolotin V, Frank R, et al. Characterization of a highly immunogenic Mycoplasma hyopneumoniae lipoprotein Mhp366 identified by peptide-spot array. Vet Microbiol, 2010, 142(3/4): [9] Simionatto S, Marchioro SB, Galli V, et al. Immunological characterization of Mycoplasma hyopneumoniae recombinant proteins. Comp Immunol Microbiol Infect Dis, 2012, 35(2): [10] Lu XM, Feng ZX, Liu MJ, et al. Establishment of a nested PCR assay for detection of Mycoplasma hyopneumoniae. Jiangsu J Agric Sci, 2010, 26(1): (in Chinese).,,,. PCR., 2010, 26(1): [11] Ding HL, Pan J, Zhang WJ, et al. Helicobacter pylori cagt protein: expression, purification and immunogenicity. J Southwest Univ: Nat Sci Ed, 2010, 32(4): (in Chinese).,,,. cagt. :, 2010, 32(4): [12] Yamao F, Muto A, Kawauchi Y, et al. UGA is read as tryptophan in Mycoplasma capricolum. Proc Natl Acad Sci USA, 1985, 82(8): [13] Sharma J, Zhong Y, Dong F, et al. Profiling of human antibody responses to Chlamydia trachomatis urogenital tract infection using microplates arrayed with 156 chlamydial fusion proteins. Infect Immun, 2006, 74(3): [14] Sharon J, Rynkiewicz MJ, Lu Z, et al. Discovery of protective B-cell epitopes for development of antimicrobial vaccines and antibody therapeutics. Immunology, 2014, 142(1): [15] Conceição FR, Moreira AN, Dellagostin OA. 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. Vaccine, 2006, 24(29/30): [16] Chen YL, Wang SN, Yang WJ, et al. Expression and immunogenicity of Mycoplasma hyopneumoniae heat shock protein antigen P42 by DNA vaccination. Infect Immun, 2003, 71(3): [17] Galli V, Simionatto S, Marchioroa SB, et al. Immunisation of mice with Mycoplasma hyopneumoniae antigens P37, P42, P46 and P95 delivered as recombinant subunit or DNA vaccines. Vaccine, 2012, 31(1): [18] Marchioro SB, Simionatto S, Galli V, et al. Production and characterization of recombinant transmembrane proteins from Mycoplasma hyopneumoniae. Vet Microbiol, 2012, 155(1):

10 周瑶琴等 / 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 53 [19] Li Z, Chen C, Chen D, et al. Characterization of fifty putative inclusion membrane proteins encoded in the Chlamydia trachomatis genome. Infect Immun, 2008, 76(6): [20] Wang J, Zhang Y, Lu C, et al. A genome-wide profiling of the humoral immune response to Chlamydia trachomatis infection reveals vaccine candidate antigens expressed in humans. J Immunol, 2010, 185(3): [21] Wang J, Chen L, Chen F, et al. A chlamydial type III-secreted effector protein (Tarp) is predominantly recognized by antibodies from humans infected with Chlamydia trachomatis and induces protective immunity against upper genital tract pathologies in mice. Vaccine, 2009, 27(22): [22] Luo J, Liu G, Zhong Y, et al. Characterization of hypothetical proteins Cpn0146, 0147, 0284 & 0285 that are predicted to be in the Chlamydia pneumoniae inclusion membrane. BMC Microbiol, 2007, 7: 38. [23] Zeng H, Gong S, Hou S, et al. Identification of antigen-specific antibody responses associated with upper genital tract pathology in mice infected with Chlamydia muridarum. Infect Immun, 2012, 80(3): [24] Zeng H, Hou S, Gong S, et al. Mapping immunodominant antigens and H-2-linked antibody responses in mice urogenitally infected with Chlamydia muridarum. Microbes Infect, 2012, 14(7-8): [25] Pendarvis K, Padula MP, Tacchi JL, et al. Proteogenomic mapping of Mycoplasma hyopneumoniae virulent strain 232. BMC Genomics, 2014, 15: 576. ( 本文责编郝丽芳 ) cjb@im.ac.cn

标题

第 39 卷第 6 期 Vol. 39 No. 6 摇摇摇摇中南医学科学杂志 Journal of Medical Science in Central South China 2011 年 11 月 Nov. 2011 沙眼衣原体 T 细胞抗原 CT143 原核表达纯化及免疫活性鉴定陆春雪1,吴移谋1,彭摇波2,刘良专1,蔡恒玲1,李忠玉1 (1. 南华大学微生物与免疫学教研室,湖南