摘要
重链抗体的可变区VHH是目前天然存在的具有完整功能的最小抗体分子片段,该抗体在基础研究、药物开发等领域应用前景极为广阔。为了构建双峰驼天然单域抗体库并对其进行初步鉴定,从5峰未经免疫的健康骆驼全血中分离淋巴细胞,提取其总RNA,之后反转录为cDNA,利用PCR技术扩增全套编码重链抗体可变区的基因序列,将经过回收并酶切纯化的VHH序列克隆到噬菌体载体上,电转化大肠杆菌TG1感受态细胞,共转化343次。结果表明,构建的双峰驼天然单域抗体库库容为4.4×10~7,转化阳性率为75%,多样性良好,体现为CDR3区碱基序列及长度存在显著差异。成功地构建了双峰驼天然单域抗体库,为今后从中筛选出具有应用价值的特异性单域抗体奠定了基础。
Variable domain of heavy chain antibody （VHH） is currently the smallest natural antibody molecule fragment with complete function which has a broad application prospect in the fields of basic research and drug development. The aim of the present study was to construct a natural single domain antibody library of Bactrian camel and to preliminarily identify the established antibody library. Total RNA was extracted from peripheral blood lymphocytes of five non-immunized healthy Bactrian camels, then reversely transcribed to cDNA, and amplified by PCR to obtain the gene which encoding the camel antibody variable region. By ligation with the phage vector, the recombinant phage was electroporated into competent cells of E. coli TG1 to construct a natural single domain antibody library of Bactrian camel. The results demonstrated that the size of the constructed library was 4.4 × 107 and the transformation positive rate was 75%; the variations in sequence and length of CDR3 region indicated the good diversity of the constructed library. A natural single domain antibody library of Bactrian camel is successfully constructed in this study, which will be fundamental for the valuable single domain antibody screening for specific antigens.
出处
《畜牧与饲料科学》
2017年第5期1-7,共7页
Animal Husbandry and Feed Science
基金
内蒙古自治区自然科学基金面上项目(2013MS0410)
内蒙古自治区高等学校科学研究项目(NJZY069)
国家自然科学基金项目(81660297)
关键词
双峰驼
天然抗体库
单域抗体
噬菌体展示技术
Bactrian camel
natural antibody library
single domain antibody
phage display technique
