基于单个外周血浆细胞技术筛选MRSA人源性单克隆抗体的实验研究
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摘要
目的建立耐甲氧西林金黄色葡萄球菌(meticillin-resistant Staphylococcus aureus,MRSA)人源性单克隆抗体筛选方法。方法从52份重组MRSA多价亚单位疫苗的Ⅰ期临床研究受试者血液样本中筛选出抗体滴度高的血样,利用流式细胞术分选其外周血单个核细胞(peripheral blood mononuclear cell,PBMC)中浆细胞,以mRNA为模板PCR扩增抗体重链、轻链可变区基因,通过重叠延伸PCR分别将将抗体重链、轻链可变区基因与CMV启动子、人恒定区及多聚A尾连接,构建成单克隆抗体线性表达载体,PEI瞬时转染293T细胞进行抗体表达。结果流式细胞术分选出1 120个浆细胞,筛选出了39个针对MRSA 5种保护性抗原α-溶血素(Hla)、铁离子表面决定蛋白B(Isd B)、金葡菌蛋白A(SpA 5)、肠毒素B(SEB)及锰离子结合蛋白C(Mnt C)的人源性单克隆抗体,ELISA结果显示抗体能与抗原蛋白结合。结论通过单个外周血浆细胞技术,成功筛选出了能够与抗原结合的MRSA人源性单克隆抗体,为研究MRSA治疗性人源性抗体奠定了良好基础。
This study was performed to establish a strategy for sorting human monoclonal antibodies against Meticillin-resistant Staphylococcus aureus(MRSA). The antibody titers of 52 blood samples from the volunteer immunized with recombinant MRSA multivalent subunit vaccine were detected, of which the samples with high antibody titers were used to sort single plasma cells. The immunoglobulin(Ig) heavy-and light-chain variable regions(VH and VL) were amplified by PCR, and then assembled with constant region, CMV promoter and poly(A) by overlapping PCR, constructing linear Ig expression cassettes. The Ig expression cassettes were transfected into 293 T cells for Ig expression. Data showed that total of 1 120 single plasma cells were sorted and 39 human monoclonal antibodies were specifically binding to MRSA antigens Hla, Isd B, SpA 5, SEB and Mnt C. In conclusion, this strategy could successfully sort out monoclonal antibodies performing great specificity and binding activity, and could be a basement for therapeutic human antibodies against MRSA.
This study was performed to establish a strategy for sorting human monoclonal antibodies against Meticillin-resistant Staphylococcus aureus(MRSA). The antibody titers of 52 blood samples from the volunteer immunized with recombinant MRSA multivalent subunit vaccine were detected, of which the samples with high antibody titers were used to sort single plasma cells. The immunoglobulin(Ig) heavy-and light-chain variable regions(VH and VL) were amplified by PCR, and then assembled with constant region, CMV promoter and poly(A) by overlapping PCR, constructing linear Ig expression cassettes. The Ig expression cassettes were transfected into 293 T cells for Ig expression. Data showed that total of 1 120 single plasma cells were sorted and 39 human monoclonal antibodies were specifically binding to MRSA antigens Hla, Isd B, SpA 5, SEB and Mnt C. In conclusion, this strategy could successfully sort out monoclonal antibodies performing great specificity and binding activity, and could be a basement for therapeutic human antibodies against MRSA.
引文
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[24]Varshney AK,Wang X,Cook E,et al.Generation,characterization,and epitope mapping of neutralizing and protective monoclonal antibodies against staphylococcal enterotoxin B-induced lethal shock[J].J Biol Chem,2011,286(11):9737-9747.
[25]Kim HK,Emolo C,Dedent AC,et al.Protein A-specific monoclonal antibodies and prevention of Staphylococcus aureus disease in mice[J].Infect Immun,2012,80(10):3460-3470.
[2]Molton JS,Tambyah PA,Ang BSP,et al.The global spread of healthcare associated multidrug-resistant bacteria:a perspective from Asia[J].Clin Infect Dis,2013.56(9):1310-1318.
[3]周恒,杨峰,章金勇,等.重组金黄色葡萄球菌疫苗免疫血清中功能性抗体OPK检测方法的建立[J].免疫学杂志,2018,34(3):252-258.
[4]Oganesyan V,Peng L,Damschroder MM,et al.Mechanisms of neutralization of ahuman anti-alpha-toxin antibody[J].J Biol Chem,2014,289(43):29874-29880.
[5]Miethke T.T cell-mediated lethal shock triggered inmice by the superantigen staphylococcal enterotoxin B:critical role of tumor necrosis factor[J].J Exp Med,1992,175(1):91-98.
[6]Chuang YY,Huang YC.Molecular epidemiology of community-associated meticillin-resistant Staphylococcus aureus in Asia[J].Lancet Infect Dis,2013,13(8):698-708.
[7]Hilliard JJ,Datta V,Tkaczyk C,et al.Anti-alpha-toxin monoclonal antibody and antibiotic combination therapy improves disease outcome and accelerates healing in a Staphylococcus aureus dermonecrosis model[J].Antimicrob Agents Chemother,2015,59(1):299-309.
[8]Bagnoli F,Bertholet S,Grandi G.Inferring reasons for the failure of Staphylococcus aureus vaccines in clinical trials[J].Front Cell Infect Microbiol,2012,2:16.
[9]Mora M,Bensi G,Capo S,et al.Group A Streptococcus produce pilus-like structures containing protective antigens and lancefield T antigens[J].Proc Natl Acad Sci U S A,2005,102(43):15641-15646.
[10]Pader V,James EH,Painter KL,et al.The Agr quorumsensing system regulates fibronectin binding but not hemolysis in the absence of a functional electron transport chain[J].Infect Immun,2014,82(10):4337-4347.
[11]Oleksiewicz MB,Nagy G,Nagy E.Anti-bacterial monoclonal antibodies:back to the future?[J].Arch Biochem Biophys,2012,526(2):124-131.
[12]Both L,Banyard AC,van Dolleweerd C,et al.Monoclonal antibodies for prophylactic and therapeutic use against viral infections[J].Vaccine,2013,31(12):1553-1559.
[13]超级细菌疫苗--金黄色葡萄球菌疫苗已获批进入Ⅰ、Ⅱ、Ⅲ期临床试验[J].第三军医大学学报,2016,38(17):2566.
[14]蔡昌芝,左钱飞,冯强,等.耐甲氧西林金黄色葡萄球菌IsdB活性片段的克隆、表达及抗原免疫保护性研究[J].免疫学杂志,2012,28(9):759-763.
[15]Liao HX,Levesque MC,Nagel A,et al.High-throughput isolation of immunoglobulin genes from single human Bcells and expression as monoclonal antibodies[J].J Virol Methods,2009,158(1/2):171-179.
[16]Tiller T,Meffre E,Yurasov S,et al.Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning[J].J Immunol Methods,2008,329(1/2):112-124.
[17]Boshart M,Weber F,Jahn G,et al.A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus[J].Cell,1985,41(2):521-530.
[18]Burstein Y,Schechter I.Primary structures of Nterminal extra peptide segments linked to the variable and constant regions of immunoglobulin light chain precursors:implications on the organization and controlled expression of immunoglobulin genes[J].Biochemistry,1978,17(12):2392-2400.
[19]Strausberg RL,Feingold EA,Grouse LH,et al.Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences[J].Proc Natl Acad Sci U S A,2002,99(26):16899-16903.
[20]Gimmi ER,Reff ME,Deckman IC.Alterations in the premRNA topology of the bovine growth hormone polyadenylation region decrease poly(A)site efficiency[J].Nucleic Acids Res,1989,17(17):6983-6998.
[21]迟象阳,于长明,陈薇,等.单个B细胞抗体制备技术及应用[J].中国生物工程学报,2012,28(6):651-660.
[22]Foletti D,Strop P,Shaughnessy L,et al.Mechanism of action and in vivo efficacy of a human-derived antibody against Staphylococcus aureus alpha-hemolysin[J].J Mol Biol,2013,425(10):1641-1654.
[23]Gregory P,Hongxia F,Sharon S,et al.Characterization of the mechanism of protection mediated by CS-D7,a monoclonal antibody to Staphylococcus aureus iron regulated surface determinant B(IsdB)[J].Front Cell Infect Microbiol,2012,2:36.
[24]Varshney AK,Wang X,Cook E,et al.Generation,characterization,and epitope mapping of neutralizing and protective monoclonal antibodies against staphylococcal enterotoxin B-induced lethal shock[J].J Biol Chem,2011,286(11):9737-9747.
[25]Kim HK,Emolo C,Dedent AC,et al.Protein A-specific monoclonal antibodies and prevention of Staphylococcus aureus disease in mice[J].Infect Immun,2012,80(10):3460-3470.