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为研制人源抗发热伴血小板减少综合征布尼亚病毒(Severe fever with thrombocytopenia syndrome virus,SFTSV)Gn蛋白重组抗体,本研究利用噬菌体表面展示技术,以SFTSV全病毒颗粒和重组表达SFTSV-Gn蛋白为抗原,从人源抗SFTSV Fab噬菌体抗体库中筛选抗SFTSV-Gn蛋白的重组Fab抗体,通过ELISA对Fab抗体的结合特异性进行检测。将Fab抗体基因克隆入哺乳动物细胞表达载体HL51-14,瞬时转染293T细胞获得分泌表达的IgG抗体。通过ELISA、IFA和Western-blotting检测IgG抗体的结合特异性。采用亲和层析纯化IgG抗体,并用微量中和试验检测IgG抗体的中和活性。结果表明经过三轮富集筛选,以SFTSV病毒颗粒为抗原筛选出364株针对SFTS病毒核蛋白Fab抗体,没有筛选出特异性结合Gn蛋白的阳性克隆,而通过Gn蛋白筛选得到8株特异结合Gn蛋白的Fab抗体,其中5株来自Lambda库,3株来自Kappa库。ELISA、IFA和Western-blotting检测证实这8株IgG抗体均能特异性结合Gn蛋白。微量中和试验显示8株新筛抗体没有中和活性,但仍可为后续SFTSV人源单克隆抗体的研究提供借鉴和参考。
In order to develop recombinant Gn antibody against fever and thrombocytopenia syndrome virus (SFTSV), phage display technique was used in this study to express the SFTSV full-length virus and recombinant SFTSV-Gn protein As antigens, a recombinant Fab antibody against SFTSV-Gn protein was screened from human anti-SFTSV Fab phage antibody library and the binding specificity of Fab antibody was detected by ELISA. The Fab antibody gene was cloned into the mammalian cell expression vector HL51-14 and transiently transfected into 293T cells to obtain secreted IgG antibodies. The binding specificity of the IgG antibodies was tested by ELISA, IFA and Western-blotting. IgG antibodies were purified by affinity chromatography and the neutralizing activity of IgG antibodies was tested by a micro-neutralization assay. The results showed that after three rounds of enrichment and screening, 364 SF antibodies against SFTS virus nucleoprotein were screened by SFTSV virus particles, and no positive clones that specifically bind to Gn protein were screened out. Eight Gn specific binding Gn Fab antibodies, of which 5 were from the Lambda library and 3 were from the Kappa library. ELISA, IFA and Western-blotting confirmed that all 8 IgG antibodies could specifically bind Gn protein. Micro neutralization test showed that there were no neutralizing activities of the 8 new screen antibodies, but still provide reference for the further study of SFTSV human monoclonal antibodies.