人源抗狂犬病毒二硫键稳定性Fv抗体的生物学特性研究

对抗狂犬病毒scFv进行稳定性改构 ,纯化制备有活性的人源抗狂犬病毒二硫键稳定性Fv抗体片段 (dsFv) ,然后对其生物学活性进行研究。将dsFvV_H 、V_L 基因在原核表达系统pET22b(+) BL21(DE3)中表达 ,将其包涵体分别在变性缓冲液中溶解 ,稀释后加入折叠缓冲液使其折叠形成有活性的dsFv抗体片段 ,上Ni-NTA柱进行纯化。然后以其亲本scFv作为对照 ,对dsFv的亲和力、稳定性以及体外中和活性进行评价。结果显示 ,与其母本抗体scFv相比 ,改构后的抗狂犬病毒dsFv保持了对狂犬病毒Vero疫苗的特异性识别能力 ,而且其亲和力明显提高 ;抗狂犬病毒dsFv在血清和BSA中的稳定性有明显的改进 ,而且其热稳定性和抵抗尿素化学变性的能力亦大大改进 ;蚀斑减少中和实验显示 ,抗狂犬病毒dsFv抗体片段能特异中和狂犬病毒 ,阻止狂犬病毒对Vero细胞的吸附 ,抑制狂犬病毒对靶细胞的感染 ,从而导致蚀斑减少甚至消失 ;scFv抗体片段仅可部分抑制蚀斑的形成 ,但不能全部抑制。这为进一步研究抗狂犬病毒dsFv基因工程抗体的免疫保护作用及其在临床的应用奠定了基础。

Improved Biochemical Characteristics of Human Disulfide-stabilized Fv Fragment to Rabies Virus

This study is (1) to improve the stabilization of human scFv to rabies virus; (2) to prepare active human dsFv fragment; and (3) to evaluate the biol ogical activities of dsFv. The dsFv V H and V L were separately expressed in P ET22 b(+)/BL21(DE3), solublized and combined in appropriate molar ratio in refolding solution. The resultant dsFv fragments were evaluated for its protection against rabies virus, its affinity and stability, in reference to the cognate scFv. Th e dsFv was found to bind specifically to Vero vaccine of rabies virus. Compared to the scFv, the dsFv was more stable, had higher affinity, and was able to inhi bit the infection of Rabies virus to Vero cell. This established a solid basis f or the clinical application of dsFv to rabies virus.