扬州大学学报（农业与生命科学版）2011，Vol.32Issue(2)：6-10,5.
猪CD163的分子克隆、原核表达及免疫血清制备
Molecular cloning, prokaryotic expression and antiserum preparation of porcine CD163
摘要
Abstract
Bioinformatics programs were used to predict the signal peptide, structural domains and antigenic index of porcine CD163. The first 1 511 bp cDNA was amplified from porcine alveolar macrophage using RT-PCR and then the 697 bp cDNA was amplified using PCR for sequencing and expression. The 697 bp cDNA was subcloned into the prokary-otic expression vector containing the extracellular domain (IgV) of canine cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and the fusion protein expression was induced with IPTG. 1CR mice were immunized for three times with 50 μg affinity-purified antigen and the specific antibody was titrated using indirect ELISA. The specificity of the immune serum was confirmed by Western blotting using GD163+ porcine alveolar macrophage as the antigen. Preparation of the recombinant antigen and antiserum against porcine CD163 for studying virus-host interaction of porcine reproductive and respiratory syndrome virus. The 45-297 aa region of porcine CD163 had higher hydrophilicity and antigenic index. Both RT-PCR and PCR products had expected sizes and the 697 bp cDNA had an identical amino acid sequence to the published sequence. The expressed fusion protein had an expected molecular weight of 43 ku which was present mainly as insoluble inclusion bodies. ELISA title of the immune serum was 1:300 000. Using the antiserum. An expected 120 ku protein band was detected in porcine alveolar macrophages, but not in PK-15 cells. The recombinant antigen and antiserum were useful reagents for detection of porcine CD163 expression.关键词
猪CD163 cDNA/分子克隆/原核表达/免疫血清Key words
porcine CD163 cDNA/cloning/prokaryotic expression/antiserum分类
生物科学引用本文复制引用
李秀媛,孙怀昌,宋红芹,刘文俊,张钰,陈阳,张鑫宇,夏晓莉..猪CD163的分子克隆、原核表达及免疫血清制备[J].扬州大学学报（农业与生命科学版）,2011,32(2):6-10,5.基金项目
公益性行业(农业)科研专项(200903037-07) （农业）
