严重发热伴血小板减少综合征病毒(SFTSV)人源性单链抗体文库的构建  

Construction of a human single-chain fragment antibody against severe fever with thrombocytopenia syndrome virus

作　　者：张文帅[1] 曾晓燕[1] 迟莹[1] 刘静娴[1] 焦永军[1] ZHANG Wenshuai;ZENG Xiaoyan;CHI Ying;LIU Jingxian;JIAO Yongjun(Jiangsu Provincial Center for Disease Prevention and Control,Nanjing 210009,China)

机构地区：[1]江苏省疾病预防控制中心,江苏南京210009

出　　处：《细胞与分子免疫学杂志》2018年第5期390-394,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(31570926)

摘　　要：目的构建人源性严重发热伴血小板减少综合征病毒(SFTSV)单链抗体(scFv)文库,并用SFTSV颗粒对文库进行初步筛选。方法提取8例SFTS恢复期患者外周血淋巴细胞的总RNA,逆转录为cDNA;并以此为模板PCR扩增人源抗体轻链库(Vκ和Vλ)和重链库(VH),再经过重叠PCR随机拼接成scFv基因文库,最后克隆入噬菌粒载体pComb3XSS中,将重组噬菌粒电转化感受态XL1-Blue细胞,得到SFTSV抗体文库,检测文库库容及多样性,并用SFTSV颗粒作抗原对抗体文库进行初步筛选。结果构建的人源SFTSV抗体文库的库容为2.8×10~7,测序结果表明文库多样性好,经过初步筛选获得21个克隆的人源化scFv,具有与SFTSV颗粒结合的活性。结论成功构建了人源抗SFTSV的scFv文库。Objective To construct a human phage display library against severe fever with thrombocytopenia syndrome（ SFTS） virus. Methods The total RNA was isolated from peripheral blood lymphocytes of 8 patients with SFTS and c DNA was amplified. The genes of light-chain（ Vκ and Vλ） and heavy-chain（ VH） were amplified by PCR. The sc Fv gene was linked by overlap-PCR and cloned into the vector p Comb3 XSS,and then transformed into XL1-Blue cells for the phage antibody library construction. After detecting the recombinant rate and the library repertoire,we screened the antibodies against SFTS virus by biopanning with immobilized virus antigen. Results The volume of constructed phage antibody library was 2. 8 × 10~7. The results of sequencing showed that the sequence of the contained single-chain variable fragment（ sc Fv）was different. After three rounds of panning,phage antibodies were specifically enriched. A total of 21 clones were positive against SFTS virus by phage-ELISA. Conclusion A high-capacity and diverse human sc Fv against SFTS virus has been constructed successfully.

关 键 词：严重发热伴血小板减少综合征病毒(SFTSV) 人源抗体文库 单链抗体(scFv) 

分 类 号：R392.1[医药卫生—免疫学] Q344.13[医药卫生—基础医学]