猪C型凝集素受体8A骆驼单域抗体酵母展示文库的构建

Construction of yeast surface display library for single domain antibody against porcine C-type lectin domain family 8,member A receptor

为构建猪C型凝集素受体8A（C-type lectin domain family 8，member A，CLEC8A）的单域抗体酵母展示文库，本文首先克隆出了猪CLEC8A基因编码区序列。通过SMART软件分析，鉴定出该受体胞外区基因序列，并插入原核表达载体pET28a（＋），在大肠杆菌BL21（DE3）中成功表达出了猪CLEC8A胞外区蛋白。表达的蛋白经201佐剂乳化后免疫双峰驼，间接ELISA监测抗体滴度，于六免后15d分离骆驼外周血淋巴细胞。提取总RNA，反转录成cDNA后经巢式PCR扩增获得骆驼WfH基因。纯化的VHH片段与线性化的pCTCON2载体混合后共转入酿酒酵母感受态细胞（EBY100），经细胞内同源重组，成功制备了针对猪CLEC8A受体的骆驼单域抗体酵母展示文库。经菌落计数及测序鉴定结果表明，文库大小约为1．6×10 7，文库重组率达90％，文库多样性丰富。流式细胞术初步鉴定酵母细胞表面成功展示出抗猪CLE8A的单域抗体，为后续文库筛选奠定基础。

To construct a yeast surface display library of antibody against porcine C-type leetin receptors family 8, member A （CLEC8A）,the coding region sequence of porcine CLEC8A gene were firstly cloned. The sequence of pig CLEC8A receptor extracellular domain was identified by SMART software and was successfully expressed in Escherichia coli BL21（DE3） by using pro- karyotic expression vector pET28a（＋）. Subsequently, bactrian camels were immunized six times with this expressed protein and peripheral blood lymphoeytes（PBLs） were isolated from whole blood. Then,the VHH fragment was obtained from the total RNA using nested RT-PCR techniques. The yeast display library against porcine CLEC8A was successfully constructed by cotransforming purified VHH fragment and linearized pCTCON2 vector into yeast competent cells （EBY100） via intracellular homologous recombination. Colony counting and sequencing results showed that the library size was about 1.6 ×10 7, the rate of the library reorganization was about 90 %,which indicated that this antibody library possessed high diversity. Preliminary identification by flow eytometry showed that yeast surface display libraries could correctly display VHH antibody against porcine CLECSA. These work laid the foundation for the subsequent library screening.