抗弓形虫主要表面抗原1单链抗体的筛选及鉴定

Screening and characterization of human scFv antibody against recombinant SAG1 of Toxoplasma

目的采用重组抗原从人源化单链抗体库(Human single fold scFv library)中筛选和鉴定抗弓形虫主要表面抗原1(rSAG1)的单链抗体,对其特异性的弓形虫靶向作用进行分析,为弓形虫靶向生物治疗提供靶向分子。方法以重组SAG1融合蛋白作为包被抗原对噬菌体抗体库进行3轮固相筛选,获得抗rSAG1融合蛋白的阳性克隆;用纯化的原核表达载体pET-32C的亲和标签(Trx-His-Stag)、鼠可溶性抗原和大肠杆菌抗原对上述阳性克隆进行差异筛选,获得能特异性结合rSAG1的噬菌体克隆;差异筛选获得的特异性阳性克隆转染大肠杆菌HB2151进行诱导表达和聚丙烯酰胺凝胶电泳(SDS-PAGE)检测,获得能分泌抗rSAG1单链抗体(anti-rSAG1scFv)的阳性噬菌体克隆;DNA测序分析抗rSAG1的单链抗体基因序列。用Ni2+螯合柱亲和纯化rSAG1单链抗体,并用免疫印迹技术(Westernblot)对纯化的抗rSAG1单链抗体进行免疫反应性检测;免疫组化检测rSAG1单链抗体对弓形虫速殖子的靶向作用。结果经过rSAG1抗原的固相筛选后获得24株阳性克隆,差异筛选后获得5株能特异性结合rSAG1的噬菌体克隆,其中有3株分泌性表达抗rSAG1的单链抗体,DNA测序和Genbank比对表明其为3株不同的抗弓形虫主要表面抗原1的单链抗体;West-ernblot表明亲和纯化的单链抗体能较好结合rSAG1;免疫组化显示抗rSAG1单链抗体可与弓形虫速殖子结合。结论利用噬菌体抗体库技术获得的特异性人源rSAG1单链抗体与弓形虫速殖子有较强的结合能力,这对弓形虫的靶向生物治疗具有潜在应用价值。

Objective To screen and characterize human scFv antibody against the recombinant SAG1 of Toxoplasma, and to evaluate its immunoresponse and observe its binding capacity to tachy-zoite of Toxoplasma. Methods Three rounds of screening on recombinant SAG1-coated im-munocups were runned to obtain anti-rSAG1 phage clones, and single positive clone was further selected by differentiated screening with different antigens. The specific anti-rSAG1 phage clones were transfected into E. colt HB2151, and induced for secreted expression of scFv antibody with IPTG. The expressed anti-rSAG1 scFv antibodies were purified with Ni2+ chelating HiTrap HP column. Its immunoresponse was evaluated with Western-blot, and its binding capacity to Toxoplasma tachyzoite was observed with the immunohistochemistry (IHC) technique. Results A total of 24 phage colonies were obtained after three rounds of solid selection. There were five specific anti-rSAG1 phage colonies after differentiated selection, and three of the five phage colonies secreted an-ti-rSAG1 scFv antibody. Anti-rSAGl scFv antibodies were acquired after Ni2+ column purification. The result of Western-blot showed that rSAG1 was recognized by the specific anti-rSAG1 scFv antibodies. The result of IHC verified that anti-rSAG1 scFv antibody specificially bound outer membrane of Toxoplasma tachyzoite. Conclusion The purified anti-rSAG1 scFv antibody has strong immunoresponse and binding capacity of Toxoplasma tachyzoite. It has a potential value as a targeting molecule for biological therapy of toxoplasmosis.