GⅡ.17型诺如病毒单克隆抗体的制备及鉴定
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摘要
目的制备抗GⅡ.17型诺如病毒(noroviruses,No V)单克隆抗体,并鉴定其生物学特性。方法将小鼠的骨髓瘤细胞SP2/0与经GⅡ.17 No V VLPs抗原免疫的BALB/c小鼠的脾细胞进行融合,通过替代性的中和试验筛选出阳性杂交瘤细胞,制备单克隆抗体并进行纯化,ELISA法检测单克隆抗体效价,SDS-PAGE法检测单克隆抗体纯度,BCA法检测单克隆抗体浓度,Biacore法检测单克隆抗体亲和力,亚型试剂鉴定单克隆抗体亚型,并进行各单克隆抗体与不同型NOV VLPs-唾液人类组织血型抗原(histo-blood group antigen,HBGA)的交叉中和试验,单抗与不同型NOV VLPs结合能力的Western blot鉴定。结果筛选出3株具有中和活性的细胞株,分别命名为E12、B7、H7,纯化后的3株单克隆抗体效价均在106以上,浓度分别为6.932、2.662和2.371 mg/ml,亲和力常数KD均为10-9 mol/L,单克隆抗体E12的纯度在93%以上,属于Ig G2a亚型,单克隆抗体B7和H7的纯度分别在98%以上,属于Ig G1亚型。3株单克隆抗体仅对GⅡ.17型No V具有中和活性,对其他型的No V无中和活性,且与8种抗原均发生结合反应,但E12对8种抗原的结合能力较B7、H7差。结论已成功制备出抗GⅡ.17 No V的单克隆抗体,为进一步研究该病毒的生物学特性及疫苗的开发奠定了基础。
Objective To prepare and characterize the monoclonal antibody(Mc Ab) against G Ⅱ.17 type norovirus(No V). Methods Mouse myeloma cells SP2/0 were fused with the spleen cells of BALB/c mice immunized with the virus-like particles(VLPs) of GⅡ.17 type No V, and positive hybridoma cells were screened by alternative neutralization test. The prepared Mc Ab was purified and determined for titer by ELISA, for purity by SDS-PAGE, for concentration by BCA method, for affinity by Biacore method, and analyzed for subtype by subtyping kit. Cross-neutralization test with different types of NOV VLPs-saliva histo-blood group antigen(HBGA)was performed, and the binding capacities of Mc Ab to different types of NOV VLPs were identified by Western blot. Results Three hybridoma cell strains with neutralizing ability were screened and named as E12, B7 and H7 respectively. The titers of Mc Abs secreted by the three strains were more than 106, while the concentrations were 6. 932, 2. 662 and 2. 371 mg/ml respectively, and the affinity constant KD were 10-9 mol/L. Mc Ab E12 with a purity of more than 93% was of subtype Ig G2 a. However, Mc Abs B7 and H7 with purities of more than 98% were of subtype Ig G1. The three kinds of Mc Abs showed only neutralizing activity to G Ⅱ.17 type No V while showed no neutralizing activity to other types, which showed binding to 8 kinds of antigens. However, the binding capacities of E12 to 8 kinds of antigens were lower than those of B7 and H7. Conclusion The Mc Abs against GⅡ.17 type No V were successfully prepared, which laid a foundation of further study on biological characteristics of No V and development of vaccine.
Objective To prepare and characterize the monoclonal antibody(Mc Ab) against G Ⅱ.17 type norovirus(No V). Methods Mouse myeloma cells SP2/0 were fused with the spleen cells of BALB/c mice immunized with the virus-like particles(VLPs) of GⅡ.17 type No V, and positive hybridoma cells were screened by alternative neutralization test. The prepared Mc Ab was purified and determined for titer by ELISA, for purity by SDS-PAGE, for concentration by BCA method, for affinity by Biacore method, and analyzed for subtype by subtyping kit. Cross-neutralization test with different types of NOV VLPs-saliva histo-blood group antigen(HBGA)was performed, and the binding capacities of Mc Ab to different types of NOV VLPs were identified by Western blot. Results Three hybridoma cell strains with neutralizing ability were screened and named as E12, B7 and H7 respectively. The titers of Mc Abs secreted by the three strains were more than 106, while the concentrations were 6. 932, 2. 662 and 2. 371 mg/ml respectively, and the affinity constant KD were 10-9 mol/L. Mc Ab E12 with a purity of more than 93% was of subtype Ig G2 a. However, Mc Abs B7 and H7 with purities of more than 98% were of subtype Ig G1. The three kinds of Mc Abs showed only neutralizing activity to G Ⅱ.17 type No V while showed no neutralizing activity to other types, which showed binding to 8 kinds of antigens. However, the binding capacities of E12 to 8 kinds of antigens were lower than those of B7 and H7. Conclusion The Mc Abs against GⅡ.17 type No V were successfully prepared, which laid a foundation of further study on biological characteristics of No V and development of vaccine.
引文
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[11]沈家瑜,徐德顺,韩建康.诺如病毒检测技术研究应用进展[J].中国卫生检验杂志,2014,24(13):1974-1976.
[12]FENG W H,XIAO Y.Etiological analysis of a GⅡnorovirus(No V)coinfection outbreak[J].Chin J Health Lab Tec,2014,24(11):1573-1575,1578.(in Chinese)冯微宏,肖勇.一起GⅡ型诺如病毒混合感染疫情的病原鉴定及分析[J].中国卫生检验杂志,2014,24(11):1573-1575,1578.
[13]YAO D,CHEN J F,YE W,et al.Etiology and genotype features analysis of an acute gastroenteritis outbreak associated with Norovirus GⅡ.17[J].Chin J Zoon,2016,32(7):641-643,650.(in Chinese)姚栋,陈静芳,叶文,等.一起诺如病毒GⅡ.17型引起的急性胃肠炎病原学诊断及基因特征分析[J].中国人兽共患病学报,2016,32(7):641-643,650.
[2]XUE L,WU Q,CAI W,et al.Molecular characterization of new emerging GII.17 norovirus strains from South China[J].Infect Genet Evolut,2016,40(6):1-7.
[3]ROBILOTTI E,DERESINSKI S,PINSKY B A.Norovirus[J].Clin Microbiol Rev,2015,28(1):134-164.
[4]PARRA G I,GREEN K Y.Genome of emerging norovirus GII.17,United States,2014[J].Emerg Infect Dis,2015,21(8):1477-1479.
[5]WANG H,WANG Q,ZHAO J,et al.Complete nucleotide sequence analysis of the norovirus GII.17:A newly emerging and dominant variant in China,2015[J].Infect Genet Evolut,2016,38(1):47-53.
[6]MARTELLA V,DECARO N,LORUSSO E,et al.Genetic heterogeneity and recombination in canine noroviruses[J].JVirol,2009,83(21):11391-11396.
[7]LINDESMITH L C,DEBBINK K,SWANSTROM J,et al.Monoclonal antibody-based antigenic mapping of norovirus GII.4-2002[J].J Virol,2012,86(2):873-883.
[8]DONALDSON E F,LINDESMITH L C,LOBUE A D,et al.Viral shape-shifting:norovirus evasion of the human immune system[J].Nat Rev Microbiol,2010,8(3):231-241.
[9]RAVN V,DABELSTEEN E.Tissue distribution of histo-blood group antigens[J].APMIS.2000,108(1):1-28.
[10]HUANG P,FARKAS T,ZHONG W,et al.Norovirus and histo-blood group antigens:demonstration of a wide spectrum of strain specificities and classification of two major binding groups among multiple binding patterns[J].J Virol,2005,79(11):6714-6722.
[11]沈家瑜,徐德顺,韩建康.诺如病毒检测技术研究应用进展[J].中国卫生检验杂志,2014,24(13):1974-1976.
[12]FENG W H,XIAO Y.Etiological analysis of a GⅡnorovirus(No V)coinfection outbreak[J].Chin J Health Lab Tec,2014,24(11):1573-1575,1578.(in Chinese)冯微宏,肖勇.一起GⅡ型诺如病毒混合感染疫情的病原鉴定及分析[J].中国卫生检验杂志,2014,24(11):1573-1575,1578.
[13]YAO D,CHEN J F,YE W,et al.Etiology and genotype features analysis of an acute gastroenteritis outbreak associated with Norovirus GⅡ.17[J].Chin J Zoon,2016,32(7):641-643,650.(in Chinese)姚栋,陈静芳,叶文,等.一起诺如病毒GⅡ.17型引起的急性胃肠炎病原学诊断及基因特征分析[J].中国人兽共患病学报,2016,32(7):641-643,650.