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

作　　者：李秀媛[1] 孙怀昌[1] 宋红芹[1] 刘文俊[1] 张钰[1] 陈阳[1] 张鑫宇[1] 夏晓莉[1] 

机构地区：[1]扬州大学兽医学院,江苏扬州225009

出　　处：《扬州大学学报（农业与生命科学版）》2011年第2期6-10,共5页Journal of Yangzhou University：Agricultural and Life Science Edition

基　　金：公益性行业(农业)科研专项(200903037-07)

摘　　要：采用生物信息学软件分析猪CD163结构,用RT-PCR从猪巨噬细胞RNA中扩增猪CD163前1 511 bp cDNA片段,再用PCR扩增抗原指数较高的697 bp cDNA片段,将其克隆入质粒载体进行序列测定;将cDNA片段克隆入含细胞毒性T细胞相关抗原4（CTLA-4）胞外区（IgV）编码序列的原核表达载体,用IPTG诱导重组菌表达;在变性条件下用镍亲和柱纯化融合蛋白,以每只50μg蛋白的剂量免疫小鼠3次,间接ELISA检测抗体效价;以表达CD163的猪巨噬细胞和不表达CD163的PK-15细胞膜为抗原,用Western blotting验证免疫血清的特异性,以获得猪CD163重组抗原和免疫血清,为猪繁殖与呼吸综合征病毒（PRRSV）感染机制研究提供检测试剂。结果表明：猪CD163的第45~297位氨基酸抗原指数较高,不含疏水性氨基酸集中区;RT-PCR扩增产物的大小与预期结果相符,与发表序列的核苷酸序列同源性为99.5%,氨基酸序列同源性为100%;重组大肠杆菌表达的CTLA-4 IgV-CD163s融合蛋白为预期的43 ku,主要以包涵体形式存在,通过亲和层析和尿素变性/复性获得了可溶性纯化蛋白;免疫小鼠血清的ELISA抗体效价为1∶300 000,能在CD163＋细胞中检测到预期的120 ku蛋白条带,而在CD163-细胞中未检测到相应的蛋白条带。结果提示获得的猪CD163重组抗原和免疫血清可用于PRRSV感染检测。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 prokaryotic 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.ICR 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 CD163＋ 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CD163cDNA 分子克隆 原核表达 免疫血清 

分 类 号：Q785[生物学—分子生物学]