鼠源性抗雄性特异性抗原噬菌体Fab抗体库的构建  

Construction of Phage Display Library of Fab Antibodies Repertoies of Mouse Male Specific Antigen

作　　者：王乃东[1] 薛立群[1] 许道军[1] 袁安文[1] 邓治邦[1] 

机构地区：[1]湖南农业大学动物医学院

出　　处：《畜牧兽医学报》2007年第4期344-351,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

摘　　要：利用雄鼠脾细胞免疫6-7周龄C57BL/6雌鼠,加强免疫后取脾细胞,TRIZOL法提取细胞总RNA,并逆转录合成cDNA第一条链。以其为模板,利用特异性Ig基因的引物PCR扩增出重链Fd片段和κ链基因,并将扩增出基因重组到噬质粒载体pComb3中,重组噬质粒转化大肠杆菌XL1-Blue中,分别构建κ链基因库和抗体Fab段基因库。分别经蓝白斑筛选,计算转化效率,通过PCR反应和双酶切反应鉴定抗体库的重组率,轻链、重链Fd段基因的重组率均为80%。抗体Fab段基因库经过辅助噬菌体VCSM13超感染,成功构建了鼠源性噬菌体Fab抗体库,并测定噬菌体抗体库的库容和滴度,结果分别为1.5×10^7,3.2×10^11pfu/mL。对噬菌体抗体库的18个克隆进行ELISA分析,结果显示以雄鼠脾细胞作为抗原时,有9个克隆含有雄性特异性的噬菌体抗体,而以雄鼠睾丸细胞作为抗原时,有8个克隆与睾丸细胞呈现结合活性。噬菌体抗体库的构建为雄性特异性抗原的鼠源性噬菌体抗体Fab片段的筛选及其应用、雄性特异性抗原和抗体功能性分子基础的研究奠定了坚实基础。Isogenic C57BL/6 female mice at 6-7 weeks of age were inoculated with male spleen cells from the same strain. RNA was isolated from immunized female mice spleen cells using TRIZOL method. The first-strand cDNA was reverse transcribed from total RNA using an oligo （dT）18 primer, the κ light-chain and the Fd （Vh-Ch1） regions of heavy-chain products were amplified from the cDNA template by polymerase chain reaction （PCR） with specific primers. To generate the pComb3-κ and pComb3- κ-Fd library, κ light chain and Fd gene fragments were inserted into phagemid vector pComb3 respectively, then transformed into E. coli strain of XL1- Blue competent cells, the transformants were plated for a blue-white color screening. The recombination rates of κ light chains and Fd fragments were 80 % which was confirmed by PCR and endonuclease digestions. A mouse combinatorial Fab library was constructed by the help phage VC- SM13 infection, which gave 1.5 × 10^7 colonies with the titer of 3.2 × 10^11pfu/mL. The presence of mouse Fab on the phage surface was determined by ELISA. 9 of 18 strains had specific binding capacity to male spleen cells, 8 of 18 strains had specific binding capacity to testicle cells. The mouse Fab antibody library could be used for selection of H-Y antigen binding Fab phage. These new molecules are potentially valuable tools in the study for molecular characteristics and functional analysis of H-Y antigen and antibody, the application of embryo sexing in mammals.

关 键 词：小鼠 H—Y抗原 噬菌体展示技术 抗体库 

分 类 号：Q782[生物学—分子生物学]