牛病毒性腹泻病毒纳米抗体文库的构建与鉴定  

Construction and Characterization of a Nanobody Library against BVDV

作　　者：张国奇[1] 李尤简 肖红冉[2] 黄美玲[1] 杨霞[2] 陈创夫[2] 盛金良[2] 

机构地区：[1]石河子大学生命科学学院,新疆石河子832000 [2]石河子大学动物科技学院,新疆石河子832000

出　　处：《西北农业学报》2015年第3期20-25,共6页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：国家国际科技合作专项(2013DFR30970)

摘　　要：通过构建牛病毒性腹泻病毒(BVDV)驼源重链抗体文库,并鉴定抗体文库的多样性,为筛选抗BVDV特异性重链抗体奠定基础。利用灭活的BVDV免疫双峰驼,多次免疫后测定血清抗体滴度,采集免疫后的血液并分离其外周淋巴细胞,抽提RNA,用RT-PCR法扩增重链抗体可变区(VHH)片段;将其克隆至载体pCANTAB5E,电转大肠杆菌TG1获得VHH抗体基因库,检测抗体库库容量,随机挑取克隆测序验证抗体文库多样性。结果表明,所构建的抗体文库的库容量为4.32×105;测序和聚类分析表明,抗体文库多样性丰富。利用辅助噬菌体救援后,得到噬菌体展示文库,测定滴度达1.3×1012 cfu/mL。A camelid variable domain of heavy chain（VHH）antibody library was constructed and the diversity of antibody library was identified for the future screening of VHH antibodies against BVDV.The camelid species（Camelus bactrianus）was selected for immunization with inactivated BVDV.After several immunized titer of serum antibodies determined.Camelus bactrianus peripheral blood mononuclear lymphocytes（PBMLs）were isolated from whole blood,total RNA was extracted,VHH were amplified by RT-PCR,PCR products were then purified and inserted into vector pCANTAB 5E,and then electroporated into E.coli TG1 cells to obtain the VHH antibody gene library.The library capacity and diversity of VHH antibody gene library was determined by sequencing analysis.The result showed that the constructed antibody library had 4.32×10^5 capacity.The sequencing and cluster analysis indicated that this antibody library possessed high diversity.After helper rescue,the phaged display library was generated with a titre up to 1.3×10^12 cfu/mL.In conclusion,a diversified singledomain antibody phage library has been constructed,which provides a platform for VHH antibody preparation against BVDV.

关 键 词：牛病毒性腹泻病毒 噬菌体抗体文库 单域抗体 VHH 

分 类 号：S852.653[农业科学—基础兽医学]