Construction and immunogenicity of a prokaryotic expression strain of <i>Staphylococcus aureus</i> fibronectin binding protein A r10-11 truncated fusion protein _West China Medical Journal

Authors：

YANG Xijing ¹ , CHEN Xiaoting ¹ , LIU Daolong ² , YANG Yang ¹ ,  YANG Guang ¹

1. Animal Experiment Center, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China;
2. College of Life Sciences, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang 163319, P. R. China;

Corresponding author：

YANG Guang, Email: 16399798@qq.com

Keywords：

Staphylococcus aureus; Fibronectin binding protein A r10-11; Immunogenicity

DOI：

10.7507/1002-0179.201811035

Video：

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ObjectiveTo construct a prokaryotic expression strain of Staphylococcus aureus fibronectin binding protein A (FnBPA) r10-11 truncated fusion protein, and explore the immunogenicity of FnBPAr10-11. MethodsPloymerase chain reaction (PCR) amplification was carried out from the whole genome sequence of Staphylococcus aureus Newman strain by recombinant PCR technique. The amplified product was purified and transformed into Escherichia coli DH5α for cloning. The recombinant plasmid was extracted and identified by double enzyme digestion. The recovered fragment was ligated into the pET-32a plasmid and transformed into Escherichia coli BL21 (DE3) for prokaryotic expression. The FnBPAr10-11 was purified by HIS protein purification column, identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and used to immunize mice, and the mice were divided into phosphate buffered saline (PBS) group, FnBPA group, and FnBPAr10-11 group. The serum levels of immunoglobulin G (IgG) and cytokines, and the immune protection rate of the mice were detected. ResultsSDS-PAGE result showed that the relative molecular mass of the protein was about 33.1×10³. The titers of IgG antibody in FnBPAr10-11 group and FnBPA group reached 1∶128 000, and were significantly different compared with PBS group (P<0.05). The cytokine level in FnBPAr10-11 group was not significantly different compared with that in FnBPA group, and they were extremely significant (P<0.01) compared with that in PBS group. The immuno-protective effect of the FnBPAr10-11 group was over 50%. ConclusionsThe prokaryotic expression strain of Staphylococcus aureu FnBPAr10-11 truncated fusion protein was successfully constructed. The truncated protein has good immunogenicity.

Citation： YANG Xijing, CHEN Xiaoting, LIU Daolong, YANG Yang, YANG Guang. Construction and immunogenicity of a prokaryotic expression strain of Staphylococcus aureus fibronectin binding protein A r10-11 truncated fusion protein . West China Medical Journal, 2018, 33(12): 1519-1525. doi: 10.7507/1002-0179.201811035 Copy

1.	Rennermalm A, Li YH, Bohaufs L, et al. Antibodies against a truncated Staphylococcus aureus fibronectin-binding protein protect against dissemination of infection in the rat. Vaccine, 2001, 19(25-26): 3376-3383.
2.	Kim HK, DeDent A, Cheng AG, et al. IsdA and IsdB antibodies protect mice against Staphylococcus aureus abscess formation and lethal challenge. Vaccine, 2010, 28(38): 6382-6392.
3.	马保臣, 董玉兰, 柴同杰. 奶牛乳腺炎金黄色葡萄球菌 FnBPA 蛋白免疫研究. 中国兽医杂志, 2018, 54(6): 49-52.
4.	Brown AF, Leech JM, Rogers TR, et al. Staphylococcus aureus colonization: modulation of host immune response and impact on human vaccine design. Front Immunol, 2014, 8(4): 507-513.
5.	Eldridge JH, Staas JK, Meulbroek JA, et al. Biodegradable and biocompatible poly (DL-lacride-co-glycolide) microspheres as an adjuvant for staphylococcal enterotoxin B toxod which enhance the level of toxin-neutralizing antibodies. Infect Immun, 1991, 59(9): 2978-2986.
6.	David MZ, Daum RS. Community-associated methicillin-resistant Staphylococcus aureus: epidemiology and clinical consequences of an emerging epidemic. Clin Microbiol Rev, 2010, 23(3): 616-687.
7.	Keane FM, Loughman A, Valtulina V, et al. Fibrinogen and elastin bind to the same region within the A domain of fibronectin binding protein A, an MSCRAMM of Staphylococcus aureus. Mol Microbiol, 2007, 63(3): 711-723.
8.	McDevitt D, Francois P, Vaudaux P, et al. Identification of the ligand-binding domain of the surface-located fibrinogen receptor (clumping factor) of Staphylococcus aureus. Mol Microbiol, 1995, 16(5): 895-907.
9.	O’Seaghdha M, van Schooten CJ, Kerrigan SW, et al. Staphylococcus aureus protein A binding to von Willebrand factor A1 domain is mediated by conserved IgG binding regions. FEBS J, 2006, 273(21): 4831-4841.
10.	Fitzgerald JR, Loughman A, Keane F, et al. Fibronectin-binding proteins of Staphylococcus aureus mediate activation of human platelets via fibrinogen and fibronectin bridges to integrin GPⅡb/Ⅲa and IgG binding to the Fcgamma RⅡa receptor. Mol Microbiol, 2006, 59(1): 212-230.
11.	Schwarz-Linek U, Werner JM, Pickford AR, et al. Pathogenic bacteria attach to human fibronectin through a tandemβ-zipper. Nature, 2003, 423(6936): 177-181.
12.	Fowler VG, Miro JM, Hoen B, et al. Staphylococcus aureus endocarditis: a consequence of medical progress. JAMA, 2005, 293(24): 3012-3021.
13.	Zuo Qianfei, Cai Changzhi, Ding Honglei, et al. Identification of the immunodominant regions of Staphylococcus aureus fibronectin-binding protein A. PLoS One, 2014, 9(4): e95338.
14.	Pilka ES, Werner JM, Schwarz-Linek U, et al. Structural insight into binding of Staphylococcus aureus to human fibronectin. FEBS Lett, 2006, 580(1): 273-277.
15.	Carratalà J, Rosón B, Fernández-Sabé N, et al. Factors associated with complications and mortality in adult patients hospitalized for infectious cellulitis. Eur J Clin Microbiol Infect Dis, 2003, 22(3): 151-157.
16.	左钱飞, 吴翼, 董衍东, 等. 耐甲氧西林金黄色葡萄球菌 (MRSA) 重组亚单位疫苗 FnBPA 的免疫保护作用研究. 免疫学杂志, 2012, 28(10): 837-841.
17.	易鸳鸯, 马陈, 苏艳. 牛乳源金黄色葡萄球菌 ClfA 与 FnBPA-A 重组质粒小鼠免疫保护效果分析. 新疆农业大学学报, 2017, 40(1): 25-31.
18.	Foster TJ. The remarkably muhifunctional flbronectin binding proteins of Staphylococcus aureus. Eur J Clin Microbiol Infect Dis, 2016, 35(12): 1923-1931.

1. Rennermalm A, Li YH, Bohaufs L, et al. Antibodies against a truncated Staphylococcus aureus fibronectin-binding protein protect against dissemination of infection in the rat. Vaccine, 2001, 19(25-26): 3376-3383.
2. Kim HK, DeDent A, Cheng AG, et al. IsdA and IsdB antibodies protect mice against Staphylococcus aureus abscess formation and lethal challenge. Vaccine, 2010, 28(38): 6382-6392.
3. 马保臣, 董玉兰, 柴同杰. 奶牛乳腺炎金黄色葡萄球菌 FnBPA 蛋白免疫研究. 中国兽医杂志, 2018, 54(6): 49-52.
4. Brown AF, Leech JM, Rogers TR, et al. Staphylococcus aureus colonization: modulation of host immune response and impact on human vaccine design. Front Immunol, 2014, 8(4): 507-513.
5. Eldridge JH, Staas JK, Meulbroek JA, et al. Biodegradable and biocompatible poly (DL-lacride-co-glycolide) microspheres as an adjuvant for staphylococcal enterotoxin B toxod which enhance the level of toxin-neutralizing antibodies. Infect Immun, 1991, 59(9): 2978-2986.
6. David MZ, Daum RS. Community-associated methicillin-resistant Staphylococcus aureus: epidemiology and clinical consequences of an emerging epidemic. Clin Microbiol Rev, 2010, 23(3): 616-687.
7. Keane FM, Loughman A, Valtulina V, et al. Fibrinogen and elastin bind to the same region within the A domain of fibronectin binding protein A, an MSCRAMM of Staphylococcus aureus. Mol Microbiol, 2007, 63(3): 711-723.
8. McDevitt D, Francois P, Vaudaux P, et al. Identification of the ligand-binding domain of the surface-located fibrinogen receptor (clumping factor) of Staphylococcus aureus. Mol Microbiol, 1995, 16(5): 895-907.
9. O’Seaghdha M, van Schooten CJ, Kerrigan SW, et al. Staphylococcus aureus protein A binding to von Willebrand factor A1 domain is mediated by conserved IgG binding regions. FEBS J, 2006, 273(21): 4831-4841.
10. Fitzgerald JR, Loughman A, Keane F, et al. Fibronectin-binding proteins of Staphylococcus aureus mediate activation of human platelets via fibrinogen and fibronectin bridges to integrin GPⅡb/Ⅲa and IgG binding to the Fcgamma RⅡa receptor. Mol Microbiol, 2006, 59(1): 212-230.
11. Schwarz-Linek U, Werner JM, Pickford AR, et al. Pathogenic bacteria attach to human fibronectin through a tandemβ-zipper. Nature, 2003, 423(6936): 177-181.
12. Fowler VG, Miro JM, Hoen B, et al. Staphylococcus aureus endocarditis: a consequence of medical progress. JAMA, 2005, 293(24): 3012-3021.
13. Zuo Qianfei, Cai Changzhi, Ding Honglei, et al. Identification of the immunodominant regions of Staphylococcus aureus fibronectin-binding protein A. PLoS One, 2014, 9(4): e95338.
14. Pilka ES, Werner JM, Schwarz-Linek U, et al. Structural insight into binding of Staphylococcus aureus to human fibronectin. FEBS Lett, 2006, 580(1): 273-277.
15. Carratalà J, Rosón B, Fernández-Sabé N, et al. Factors associated with complications and mortality in adult patients hospitalized for infectious cellulitis. Eur J Clin Microbiol Infect Dis, 2003, 22(3): 151-157.
16. 左钱飞, 吴翼, 董衍东, 等. 耐甲氧西林金黄色葡萄球菌 (MRSA) 重组亚单位疫苗 FnBPA 的免疫保护作用研究. 免疫学杂志, 2012, 28(10): 837-841.
17. 易鸳鸯, 马陈, 苏艳. 牛乳源金黄色葡萄球菌 ClfA 与 FnBPA-A 重组质粒小鼠免疫保护效果分析. 新疆农业大学学报, 2017, 40(1): 25-31.
18. Foster TJ. The remarkably muhifunctional flbronectin binding proteins of Staphylococcus aureus. Eur J Clin Microbiol Infect Dis, 2016, 35(12): 1923-1931.

West China Medical Journal

Construction and immunogenicity of a prokaryotic expression strain of Staphylococcus aureus fibronectin binding protein A r10-11 truncated fusion protein

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