抗Ⅰ型鸭肝炎病毒单链抗体库的构建与筛选  

Construction and screening of ScFv library against Duck hepatitis virus type Ⅰ

作　　者：韩宜均 孙举[1] 原昆鹏 曲雪婷 栾庆东 尹燕博[1] 王建琳[1] HAN Yijun;SUN Ju;YUAN Kunpeng;QU Xueting;LUAN Qingdong;YIN Yanbo;WANG Jianlin(College of Veterinan Medicine,Qingdao Agricultural Iniversity,Qingdao 266109,China)

机构地区：[1]青岛农业大学动物医学院,山东青岛266109

出　　处：《黑龙江畜牧兽医》2021年第3期91-96,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家重点研发计划项目(2016YFD0500800)。

摘　　要：为了构建和筛选抗Ⅰ型鸭肝炎病毒(DHV-Ⅰ)单链抗体库,试验用Ⅰ型鸭肝炎疫苗免疫樱桃谷鸭,以分离的外周血淋巴细胞总RNA为模板,根据抗体保守区序列设计引物,通过RT-PCR技术扩增出鸭IgG轻链可变区和重链可变区基因,由Linker序列连接,用重叠延伸PCR(SOR-PCR)技术将其连接成单链抗体基因,将扩增的单链抗体基因导入pComb3XSS载体中构建抗DHV-Ⅰ噬菌体单链抗体库,并对该噬菌体单链抗体库进行3次吸附-洗脱-富集。结果表明:试验成功构建出库量为8.5×109pfu/mL的抗DHV-Ⅰ噬菌体单链抗体库,筛选出1株亲和力较高的抗DHV-Ⅰ噬菌体单链抗体。说明试验成功构建了鸭源抗DHV-Ⅰ噬菌体单链抗体库,筛选出1株与DHV-Ⅰ亲和力良好的鸭源噬菌体单链抗体。In order to construct and screen anti-type Ⅰ Duck hepatitis virus(DHV-Ⅰ) single-chain antibody library,type Ⅰ duck hepatitis vaccine was used in this experiment to immunize Cherry Valley duck. Using the total RNA of isolated peripheral blood lymphocytes as a template,according to the sequence of antibody conservative regions to design primers,duck IgG light chain variable region and heavy chain variable region genes were amplified by RT-PCR method,which were connected by Linker sequence. Single-chain antibody genes were amplified by overlap extension PCR(SOR-PCR) method,and was introduced into the pComb3 XSS vector to construct a phage antibody library. The anti-DHV-Ⅰ phage antibody library was subjected to adsorption-elution-enrichment three times. The results showed that an anti-DHV-Ⅰ phage antibody library with a library volume of 8.5×10~9 pfu/mL was successfully constructed,and a phage ScFv against DHV-Ⅰ with high affinity was screened. The results suggested that a duck-derived anti-DHV phage single-chain antibody library was successfully constructed in this experiment,and a duck-derived phage ScFv against DHV-Ⅰ was screened.

关 键 词：Ⅰ型鸭肝炎病毒(DHV-Ⅰ) 噬菌体抗体库 单链抗体(ScFv) 构建 筛选 纯化 

分 类 号：S852.65[农业科学—基础兽医学] S858.32[农业科学—兽医学]