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首页> 外文期刊>BMC Biotechnology >Generation of recombinant single-chain antibodies neutralizing the cytolytic activity of vaginolysin, the main virulence factor of Gardnerella vaginalis
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Generation of recombinant single-chain antibodies neutralizing the cytolytic activity of vaginolysin, the main virulence factor of Gardnerella vaginalis

机译：重组单链抗体的产生，中和阴道加德纳菌的主要毒力因子vaginolysin的溶细胞活性

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Background Gardnerella vaginalis is identified as the predominant colonist of the vaginal tract in women with bacterial vaginosis. Vaginolysin (VLY) is a protein toxin released by G. vaginalis . VLY possesses cytolytic activity and is considered as a main virulence factor of G. vaginalis . Inhibition of VLY-mediated cell lysis by antibodies may have important physiological relevance. Results Single-chain variable fragments of immunoglobulins (scFvs) were cloned from two hybridoma cell lines producing neutralizing antibodies against VLY and expressed as active proteins in E. coli . For each hybridoma, two variants of anti-VLY scFv consisting of either VL-VH or VH-VL linked with a 20 aa-long linker sequence (G4S)4 were constructed. Recovery of scFvs from inclusion bodies with subsequent purification by metal-chelate chromatography resulted in VLY-binding proteins that were predominantly monomeric. The antigen-binding activity of purified scFvs was verified by an indirect ELISA . The neutralizing activity was investigated by in vitro hemolytic assay and cytolytic assay using HeLa cell line. Calculated apparent Kd values and neutralizing potency of scFvs were in agreement with those of parental full-length antibodies. VH-VL and VL-VH variants of scFvs showed similar affinity and neutralizing potency. The anti-VLY scFvs derived from hybridoma clone 9B4 exhibited high VLY-neutralizing activity both on human erythrocytes and cervical epithelial HeLa cells. Conclusions Hybridoma -derived scFvs with VLY-binding activity were expressed in E. coli . Recombinant anti-VLY scFvs inhibited VLY-mediated cell lysis. The monovalent scFvs showed reduced affinity and neutralizing potency as compared to the respective full-length antibodies. The loss of avidity could be restored by generating scFv constructs with multivalent binding properties. Generated scFvs is the first example of recombinant single-chain antibodies with VLY-neutralizing activity produced in prokaryote expression system. G. vaginalis caused infections continue to be a world-wide problem, therefore neutralizing recombinant antibodies may provide novel therapeutic agents useful in the treatment of bacterial vaginosis and other diseases caused by G. vaginalis .
机译：背景阴道加德纳菌被确定为细菌性阴道病女性的主要阴道菌落。阴道溶素（VLY）是由阴道加德纳酵母释放的一种蛋白质毒素。 VLY具有溶细胞活性，被认为是阴道志贺菌的主要毒力因子。抗体对VLY介导的细胞裂解的抑制作用可能具有重要的生理意义。结果从两个杂交瘤细胞系中克隆了免疫球蛋白（scFvs）的单链可变片段，该细胞产生针对VLY的中和抗体，并在大肠杆菌中表达为活性蛋白。对于每种杂交瘤，两种抗VLY scFv变体由VL-VH或VH-VL组成，并与20个氨基酸长的连接子序列相连（G 4 S ）构造了 4 。从包涵体中回收scFv，随后通过金属螯合物色谱法纯化，产生主要为单体的VLY结合蛋白。通过间接ELISA验证了纯化的scFv的抗原结合活性。通过体外溶血测定和使用HeLa细胞系的细胞溶解测定来研究中和活性。计算的表观K d 值和scFv的中和力与亲本全长抗体一致。 scFv的VH-VL和VL-VH变体显示出相似的亲和力和中和力。来源于杂交瘤克隆9B4的抗VLY scFvs对人红细胞和宫颈上皮HeLa细胞均显示高VLY中和活性。结论在大肠杆菌中表达了具有VLY结合活性的杂交瘤来源的scFv。重组抗VLY scFvs抑制VLY介导的细胞裂解。与相应的全长抗体相比，单价scFv显示出降低的亲和力和中和力。亲和力的丧失可以通过产生具有多价结合特性的scFv构建体来恢复。产生的scFvs是在原核生物表达系统中产生的具有VLY中和活性的重组单链抗体的第一个实例。引起由阴道加德纳氏菌引起的感染的问题仍然是世界范围内的问题，因此中和重组抗体可以提供可用于治疗细菌性阴道病和其他由阴道加德纳尔氏菌引起的疾病的新型治疗剂。

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