摘要
CD154在抗原特异的T淋巴细胞和抗原提呈细胞的相互作用中发挥非常重要的作用。利用RT—PCR方法从三黄鸡的外周血单核细胞（PBMC）中克隆了鸡CD154基因全长cDNA，序列分析显示该基因cDNA全长819bp，可编码272个氨基酸。将克隆的鸡CD154全长cDNA克隆到原核表达载体pET-28a中获得表达质粒pET-28a—CD154，转化BL21（DE3）后未检测到特异性表达蛋白。进一步通过PCR获得去除信号肽（1～22aa）和跨膜区（23～46aa）的CD154基因片段（CD154d），并将其克隆到pQE80的6×His下游，在大肠杆菌中成功表达，获得分子量约27ku的融合表达蛋白6×His—CD154d。Western—blot试验证实表达的融合蛋白能与抗6×His的单抗发生特异性反应。同时，将CD154基因全长cDNA插入真核表达载体pEGFP-C1的EGFP基因下游，获得真核表达质粒pEGFP—CD154，转染Hela细胞，荧光显微镜观察发现EGFP—CD154融合蛋白表达在细胞膜上，证实了鸡CD154的膜定位特性。本研究为进一步研究鸡CD154的结构与功能及其免疫佐剂效应奠定了基础。
CD154 plays an important role in the interaction between antigen-specific T lympho- cytes and antigen-presenting cells. In this study, the full-length cDNA encoding chicken CD154 was amplified from isolated peripheral blood mononuclear cells （PBMC） of Sanhuang chicken using RT-PCR. Sequence analysis showed that the chicken CD154 is comprised of 819 base pairs in length and encodes a 272 amino acid （aa） protein. The full-length cDNA fragment of chicken CD154 was further subcloned into prokaryotic expression vector pET-28a, resulting in the expression plasmid pET-28a-CD154. However, no specific expressed protein was detected after transformation of BL21（DE3）. The truncated fragment of chicken CD154 （CD154d） without signal peptide （1-22 aa） and transmembrance region （23-46 aa） was cloned using PCR and further subcloned into downstream of hexahistidine sequence of pQE80. Expression of a fusion protein （6 × His-CD154d） with 27 ku molecular mass was observed in E. coli DH5a, and the result of Western-blot demonstrated that the fusion protein can be recognized by anti-6 × His monoclonal antibody. In addition, the full-length cDNA fragment of chicken CD154 was inserted into down- stream of EGFP gene of eurokaryotic expression vector pEGFP-C1, resulting in the expression plasmid pEGFP-C1-CD154. After transfection in Hela cells, the fluorescence of the fusion protein EGFP-CD154 was observed on cell membrane by fluorescent microscope, indicating that the chicken CD154 is a membranessociated protein.The present study laid a foundation for further investigating the structure and function of chicken CD154 and its adjuvant potency in chicken vaccines.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2007年第10期1032-1037,共6页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家"973"基础研究项目(2005CB523200)
国家科技支撑计划(2006BAD06A04)
关键词
鸡
CD154
克隆
表达
定位
chicken
CD154
clone
expression
location
