中国免疫学杂志2011，Vol.27Issue(1)：44-49,6.DOI:10.3969/j.issn.1000-484X.2011.01.011
抗H5N1禽流感病毒VHH抗体库的构建
Construction of phage display VHH antibody library against avian H5N1 virus from alpaca
严安 1熊慧 1王颖 2孙冰玉 1夏立亮 1吴标 2包文静 1车小燕 3孙志伟 4金维荣 5赵国屏1
作者信息
- 1. 上海人类基因组研究中心,卫生部重点疾病与健康重点实验室,上海,201203
- 2. 上海市免疫学研究所,上海交通大学医学院,上海,200025
- 3. 南方医科大学,广州,510515
- 4. 北京军事医学科学院生物工程研究所,北京,100071
- 5. 上海华冠生物芯片有限公司,上海,201203
- 折叠
摘要
Abstract
Objective: To construct phage display variable domain of heavy chain antibody library (VHH antibody library) from alpaca immunized with inactivated H5N1 vaccine for the future screening of VHH antibodies against avian H5N1 influenza virus. Methods: The camelid species (alpaca, Lama pacos ) was selected for immunization with inactivated H5N1 vaccine. Hemagglutinin inhibition (HI) assay of serum from immunized alpaca was performed against H5N1 avian influenza virus one week after the fourth inntmization. Peripheral lymphocytes were isolated for the amplification of VHH fragments by RT-PCR. PCR products were then purified and inserted into phagemid vector pCANTAB5E. The VHH antibody gene library was obtained by electroporating recombinant pCANTAB5E-VHH vectors into E. coli TG1 cells.The library capacity and diversity of VHH antibody gene library was determined by sequencing analysis. The HI assay was performed with the culture supernatant of primary phage display VHH antibody library. Results:After four rounds of immunization with inactivated H5N1 vaccine,HI antibody titer of the alpaca serum reached to 1: 2 560, which was higher than those fiom immunized mice. A first set of antibody gene library totalling 3 × l08 members were created after cloning VHH genes into a phagemid vector pCANTAB5E. The sequence of 14 members of the unselected library indicated that the camelid VHH antibody library we constructed possessed high diversity and good capacity. The supematant from the primary phage display library displayed effieient HI effect against avian H5N1 influenza virus. And the titration of our phage display VHH library reached 2.17 × l011. Conclusion: Taken together, phage display VHH antibody library from immunized alpaca is successfully constructed,which provides a platform for VHH antibody preparation against H5N1 virus. This will give light to future study on treatment and diagnosis of avian H5N1 influenza virus.关键词
H5N1/噬菌体抗体库/重链可变区抗体/禽流感病毒分类
医药卫生引用本文复制引用
严安,熊慧,王颖,孙冰玉,夏立亮,吴标,包文静,车小燕,孙志伟,金维荣,赵国屏..抗H5N1禽流感病毒VHH抗体库的构建[J].中国免疫学杂志,2011,27(1):44-49,6.
